Process for the biological production of 1,3-propanediol with high titer

ABSTRACT

The present invention provides an improved method for the biological production of 1,3-propanediol from a fermentable carbon source in a single microorganism. In one aspect of the present invention, an improved process for the conversion of glucose to 1,3-propanediol is achieved by the use of an  E. coli  transformed with the  Klebsiella pneumoniae  dha regulon genes dhaR, orfY, dhaT, orfX, orfW, dhaB1, dhaB2, dhaB3, and orfZ, all these genes arranged in the same genetic organization as found in wild type  Klebsiella pneumoniae . In another aspect of the present invention, an improved process for the production of 1,3-propanediol from glucose using a recombinant  E. coli  containing genes encoding a G3PDH, a G3P phosphatase, a dehydratase, and a dehydratase reactivation factor compared to an identical process using a recombinant  E. coli  containing genes encoding a G3PDH, a G3P phosphatase, a dehydratase, a dehydratase reactivation factor and a 1,3-propanediol oxidoreductase (dhaT). The dramatically improved process relies on the presence in  E. coli  of a gene encoding a non-specific catalytic activity sufficient to convert 3-hydroxypropionaldehyde to 1,3-propanediol.

CROSS-REFERENCE TO RELATED APPLICATION

This is a divisional of allowed U.S. patent application Ser. No.11/282,497, filed Jan. 16, 2006, which is a divisional of U.S. Pat. No.7,067,300, which is a divisional of U.S. Pat. No. 6,514,733, thedisclosures of which are hereby incorporated by reference.

FIELD OF INVENTION

This invention comprises process for the bioconversion of a fermentablecarbon source to 1,3-propanediol by a single microorganism.

BACKGROUND

1,3-Propanediol is a monomer having potential utility in the productionof polyester fibers and the manufacture of polyurethanes and cycliccompounds.

A variety of chemical routes to 1,3-propanediol are known. For exampleethylene oxide may be converted to 1,3-propanediol over a catalyst inthe presence of phosphine, water, carbon monoxide, hydrogen and an acid,by the catalytic solution phase hydration of acrolein followed byreduction, or from compounds such as glycerol, reacted in the presenceof carbon monoxide and hydrogen over catalysts having atoms from groupVIII of the periodic table. Although it is possible to generate1,3-propanediol by these methods, they are expensive and generate wastestreams containing environmental pollutants.

It has been known for over a century that 1,3-propanediol can beproduced from the fermentation of glycerol. Bacterial strains able toproduce 1,3-propanediol have been found, for example, in the groupsCitrobacter, Clostridium, Enterobacter, Ilyobacter, Klebsiella,Lactobacillus, and Pelobacter. In each case studied, glycerol isconverted to 1,3-propanediol in a two step, enzyme catalyzed reactionsequence. In the first step, a dehydratase catalyzes the conversion ofglycerol to 3-hydroxypropionaldehyde (3-HPA) and water, Equation 1. Inthe second step, 3-HPA is reduced to 1,3-propanediol by a NAD⁺-linkedoxidoreductase, Equation 2. The 1,3-propanediol is not metabolizedfurther and, as a result,

Glycerol→3-HPA+H₂O  (Equation 1)

3-HPA+NADH+H⁺→1,3-Propanediol+NAD⁺  (Equation 2)

accumulates in the media. The overall reaction consumes a reducingequivalent in the form of a cofactor, reduced β-nicotinamide adeninedinucleotide (NADH), which is oxidized to nicotinamide adeninedinucleotide (NAD⁺).

In Klebsiella pneumonia, Citrobacter freundii, and Clostridiumpasteurianum, the genes encoding the three structural subunits ofglycerol dehydratase (dhaB1-3 or dhaB, C and E) are located adjacent toa gene encoding a specific 1,3-propanediol oxidoreductase (dhaT) (seeFIG. 1). Although the genetic organization differs somewhat among thesemicroorganisms, these genes are clustered in a group which alsocomprises orfX and orfZ (genes encoding a dehydratase reactivationfactor for glycerol dehydratase), as well as orfY and orfW (genes ofunknown function). The specific 1,3-propanediol oxidoreductases (dhaTs)of these microorganisms are known to belong to the family of type IIIalcohol dehydrogenases; each exhibits a conserved iron-binding motif andhas a preference for the NAD⁺/NADH linked interconversion of1,3-propandiol and 3-HPA. However, the NAD⁺/NADH linked interconversionof 1,3-propandiol and 3-HPA is also catalyzed by alcohol dehydrogenaseswhich are not specifically linked to dehydratase enzymes (for example,horse liver and baker's yeast alcohol dehydrogenases (E.C. 1.1.1.1)),albeit with less efficient kinetic parameters. Glycerol dehydratase(E.C. 4.2.1.30) and diol[1,2-propanediol]dehydratase (E.C. 4.2.1.28) arerelated but distinct enzymes that are encoded by distinct genes. Dioldehydratase genes from Klebsiella oxytoca and Salmonella typhimurium aresimilar to glycerol dehydratase genes and are clustered in a group whichcomprises genes analogous to orfX and orfZ (Daniel et al., FEMSMicrobiol. Rev. 22, 553 (1999); Toraya and Mori, J. Biol. Chem. 274,3372 (1999); GenBank AF026270).

The production of 1,3-propanediol from glycerol is generally performedunder anaerobic conditions using glycerol as the sole carbon source andin the absence of other exogenous reducing equivalent acceptors. Underthese conditions, in e.g., strains of Citrobacter, Clostridium, andKlebsiella, a parallel pathway for glycerol operates which firstinvolves oxidation of glycerol to dihydroxyacetone (DHA) by a NAD⁺- (orNADP⁺-) linked glycerol dehydrogenase, Equation 3. The DHA, followingphosphorylation to dihydroxyacetone phosphate (DHAP) by a DHA kinase(Equation 4),

Glycerol+NAD⁺→DHA+NADH+H⁺  (Equation 3)

DHA+ATP→DHAP+ADP  (Equation 4)

becomes available for biosynthesis and for supporting ATP generation viae.g., glycolysis. In contrast to the 1,3-propanediol pathway, thispathway may provide carbon and energy to the cell and produces ratherthan consumes NADH.

In Klebsiella pneumoniae and Citrobacter freundii, the genes encodingthe functionally linked activities of glycerol dehydratase (dhaB),1,3-propanediol oxidoreductase (dhaT), glycerol dehydrogenase (dhaD),and dihydroxyacetone kinase (dhaK) are encompassed by the dha regulon.The dha regulon, in Klebsiella pneumoniae and Citrobacter freundii, alsoencompasses a gene encoding a transcriptional activator protein (dhaR).The dha regulons from Citrobacter and Klebsiella have been expressed inEscherichia coli and have been shown to convert glycerol to1,3-propanediol.

Neither the chemical nor biological methods described above for theproduction of 1,3-propanediol are well suited for industrial scaleproduction since the chemical processes are energy intensive and thebiological processes are limited to relatively low titer from theexpensive starting material, glycerol. These drawbacks could be overcomewith a method requiring low energy input and an inexpensive startingmaterial such as carbohydrates or sugars, or by increasing the metabolicefficiency of a glycerol process. Development of either method willrequire the ability to manipulate the genetic machinery responsible forthe conversion of sugars to glycerol and glycerol to 1,3-propanediol.

Biological processes for the preparation of glycerol are known. Theoverwhelming majority of glycerol producers are yeasts but somebacteria, other fungi and algae are also known. Both bacteria and yeastsproduce glycerol by converting glucose or other carbohydrates throughthe fructose-1,6-bisphosphate pathway in glycolysis or the EmbdenMeyerhof Parnas pathway, whereas, certain algae convert dissolved carbondioxide or bicarbonate in the chloroplasts into the 3-carbonintermediates of the Calvin cycle. In a series of steps, the 3-carbonintermediate, phosphoglyceric acid, is converted to glyceraldehyde3-phosphate which can be readily interconverted to its keto isomerdihydroxyacetone phosphate and ultimately to glycerol.

Specifically, the bacteria Bacillus licheniformis and Lactobacilluslycopersica synthesize glycerol, and glycerol production is found in thehalotolerant algae Dunaliella sp. and Asteromonas gracilis forprotection against high external salt concentrations. Similarly, variousosmotolerant yeasts synthesize glycerol as a protective measure. Moststrains of Saccharomyces produce some glycerol during alcoholicfermentation, and this can be increased physiologically by theapplication of osmotic stress. Earlier this century commercial glycerolproduction was achieved by the use of Saccharomyces cultures to which“steering reagents” were added such as sulfites or alkalis. Through theformation of an inactive complex, the steering agents block or inhibitthe conversion of acetaldehyde to ethanol; thus, excess reducingequivalents (NADH) are available to or “steered” towards DHAP forreduction to produce glycerol. This method is limited by the partialinhibition of yeast growth that is due to the sulfites. This limitationcan be partially overcome by the use of alkalis that create excess NADHequivalents by a different mechanism. In this practice, the alkalisinitiated a Cannizarro disproportionation to yield ethanol and aceticacid from two equivalents of acetaldehyde.

The gene encoding glycerol-3-phosphate dehydrogenase (DAR1, GPD1) hasbeen cloned and sequenced from S. diastaticus (Wang et al., J. Bact.176, 7091-7095 (1994)). The DAR1 gene was cloned into a shuttle vectorand used to transform E. coli where expression produced active enzyme.Wang et al. (supra) recognize that DAR1 is regulated by the cellularosmotic environment but do not suggest how the gene might be used toenhance 1,3-propanediol production in a recombinant microorganism.

Other glycerol-3-phosphate dehydrogenase enzymes have been isolated: forexample, sn-glycerol-3-phosphate dehydrogenase has been cloned andsequenced from Saccharomyces cerevisiae (Larason et al., Mol. Microbiol.10, 1101 (1993)) and Albertyn et al. (Mol. Cell. Biol. 14, 4135 (1994))teach the cloning of GPD1 encoding a glycerol-3-phosphate dehydrogenasefrom Saccharomyces cerevisiae. Like Wang et al. (supra), both Albertynet al. and Larason et al. recognize the osmo-sensitivity of theregulation of this gene but do not suggest how the gene might be used inthe production of 1,3-propanediol in a recombinant microorganism.

As with G3PDH, glycerol-3-phosphatase has been isolated fromSaccharomyces cerevisiae and the protein identified as being encoded bythe GPP1 and GPP2 genes (Norbeck et al., J. Biol. Chem. 271, 13875(1996)). Like the genes encoding G3PDH, it appears that GPP2 isosmosensitive.

Although a single microorganism conversion of fermentable carbon sourceother than glycerol or dihydroxyacetone to 1,3-propanediol is desirable,it has been documented that there are significant difficulties toovercome in such an endeavor. For example, Gottschalk et al. (EP 373230) teach that the growth of most strains useful for the production of1,3-propanediol, including Citrobacter freundii, Clostridiumautobutylicum, Clostridium butylicum, and Klebsiella pneumoniae, isdisturbed by the presence of a hydrogen donor such as fructose orglucose. Strains of Lactobacillus brevis and Lactobacillus buchner,which produce 1,3-propanediol in co-fermentations of glycerol andfructose or glucose, do not grow when glycerol is provided as the solecarbon source, and, although it has been shown that resting cells canmetabolize glucose or fructose, they do not produce 1,3-propanediol(Veiga DA Cunha et al., J. Bacteriol., 174, 1013 (1992)). Similarly, ithas been shown that a strain of Ilyobacter polytropus, which produces1,3-propanediol when glycerol and acetate are provided, will not produce1,3-propanediol from carbon substrates other than glycerol, includingfructose and glucose (Steib et al., Arch. Microbiol. 140, 139 (1984)).Finally, Tong et al. (Appl. Biochem. Biotech. 34, 149 (1992)) taughtthat recombinant Escherichia coli transformed with the dha regulonencoding glycerol dehydratase does not produce 1,3-propanediol fromeither glucose or xylose in the absence of exogenous glycerol.

Attempts to improve the yield of 1,3-propanediol from glycerol have beenreported where co-substrates capable of providing reducing equivalents,typically fermentable sugars, are included in the process. Improvementsin yield have been claimed for resting cells of Citrobacter freundii andKlebsiella pneumoniae. DSM 4270 co-fermenting glycerol and glucose(Gottschalk et al., supra.; and Tran-Dinh et al., DE 3734 764); but notfor growing cells of Klebsiella pneumoniae. ATCC 25955 co-fermentingglycerol and glucose, which produced no 1,3-propanediol (1-T. Tong,Ph.D. Thesis, University of Wisconsin-Madison (1992)). Increased yieldshave been reported for the cofermentation of glycerol and glucose orfructose by a recombinant Escherichia coli; however, no 1,3-propanediolis produced in the absence of glycerol (Tong et al., supra.). In thesesystems, single microorganisms use the carbohydrate as a source ofgenerating NADH while providing energy and carbon for cell maintenanceor growth. These disclosures suggest that sugars do not enter the carbonstream that produces 1,3-propanediol.

Recently, however, the conversion of carbon substrates, other thanglycerol or dihydroxyacetone, to 1,3-propanediol by a singlemicroorganism that expresses a dehydratase enzyme has been described(U.S. Pat. No. 5,686,276; WO 9821339; WO 9928480; and WO 9821341 (U.S.Pat. No. 6,013,494)). A specific deficiency in the biological processesleading to the production of 1,3-propanediol from either glycerol orglucose has been the low titer of the product achieved via fermentation;thus, an energy-intensive separation process to obtain 1,3-propanediolfrom the aqueous fermentation broth is required. Fed batch or batchfermentations of glycerol to 1,3-propanediol have led to final titers of65 g/L by Clostridium butyricum (Saint-Amans et al., BiotechnologyLetters 16, 831 (1994)), 71 g/L by Clostridium butyricum mutants(Abbad-Andaloussi et al., Appl. Environ. Microbiol. 61, 4413 (1995)), 61g/L by Klebsiella pneumoniae (Homann et al., Appl. Bicrobiol.Biotechnol. 33, 121 (1990)), and 35 g/L by Citrobacter freundii (Homannet al., supra). Fermentations of glucose to 1,3-propanediol that exceedthe titer obtained from glycerol fermentations have not yet beendisclosed.

The problem that remains to be solved is how to biologically produce1,3-propanediol, with high titer and by a single microorganism, from aninexpensive carbon substrate such as glucose or other sugars. Thebiological production of 1,3-propanediol requires glycerol as asubstrate for a two-step sequential reaction in which a dehydrataseenzyme (typically a coenzyme B₁₂-dependent dehydratase) convertsglycerol to an intermediate, 3-hydroxypropionaldehyde, which is thenreduced to 1,3-propanediol by a NADH- (or NADPH) dependentoxidoreductase. The complexity of the cofactor requirements necessitatesthe use of a whole cell catalyst for an industrial process that utilizesthis reaction sequence for the production of 1,3-propanediol.

SUMMARY OF THE INVENTION

Applicants have solved the stated problem and the present inventionprovides for bioconverting a fermentable carbon source directly to1,3-propanediol at significantly higher titer than previously obtainedand with the use of a single microorganism. Glucose is used as a modelsubstrate and E. coli is used as the model host. In one aspect of thisinvention, recombinant E. coli expressing a group of genes (comprisinggenes that encode a dehydratase activity, a dehydratase reactivationfactor, a 1,3-propanediol oxidoreductase (dhaT), a glycerol-3-phosphatedehydrogenase, and a glycerol-3-phosphatase) convert glucose to1,3-propanediol at titer that approaches that of glycerol to1,3-propanediol fermentations.

In another aspect of this invention, the elimination of the functionaldhaT gene in this recombinant E. coli results in a significantly highertiter of 1,3-propanediol from glucose. This unexpected increase in titerresults in improved economics, and thus, an improved process for theproduction of 1,3-propanediol from glucose.

Furthermore, the present invention may be generally applied to includeany carbon substrate that is readily converted to 1) glycerol, 2)dihydroxyacetone, 3) C₃ compounds at the oxidation state of glycerol(e.g., glycerol 3-phosphate), or 4) C₃ compounds at the oxidation stateof dihydroxyacetone (e.g., dihydroxyacetone phosphate or glyceraldehyde3-phosphate). The production of 1,3-propanediol in the dhaT minus strainrequires a non-specific catalytic activity that converts 3-HPA to1,3-propanediol. Identification of the enzyme(s) and/or gene(s)responsible for the non-specific catalytic activity that converts 3-HPAto 1,3-propanediol will lead to production of 1,3-propanediol in a widerange of host microorganisms with substrates from a wide range ofcarbon-containing substrates. It is also anticipated that the use ofthis non-specific catalytic activity that converts 3-HPA to1,3-propanediol will lead to an improved process for the production of1,3-propanediol from glycerol or dihydroxyacetone, by virtue of animproved titer and the resulting improved economics.

This activity has been isolated from E. coli as a nucleic acid fragmentencoding a non-specific catalytic activity for the conversion of3-hydroxypropionaldehyde to 1,3-propanediol, as set out in SEQ ID NO:58or as selected from the group consisting of:

-   -   (a) an isolated nucleic acid fragment encoding all or a        substantial portion of the amino acid sequence of SEQ ID NO:57;    -   (b) an isolated nucleic acid fragment that is substantially        similar to an isolated nucleic acid fragment encoding all or a        substantial portion of the amino acid sequence of SEQ ID NO:57;    -   (c) an isolated nucleic acid fragment encoding a polypeptide of        at least 387 amino acids having at least 80% with the amino acid        sequence of SEQ ID NO:57;    -   (d) an isolated nucleic acid fragment that hybridizes with (a)        under hybridization conditions of 0.1×SSC, 0.1% SDS, 65° C. and        washed with 2×SSC, 0.1% SDS followed by 0.1×SSC, 0.1% SDS; and    -   (d) an isolated nucleic acid fragment that is complementary to        (a), (b), (c), or (d). Alternatively, the nonspecific catalytic        activity is embodied in the polypeptide as set out in SEQ ID        NO:57.

A chimeric gene may be constructed comprising the isolated nucleic acidfragment described above operably linked to suitable regulatorysequences. This chimeric gene can be used to transform microorganismsselected from the group consisting of Citrobacter, Enterobacter,Clostridium, Klebsiella, Aerobacter, Lactobacillus, Aspergillus,Saccharomyces, Schizosaccharomyces, Zygosaccharomyces, Pichia,Kluyveromyces, Candida, Hansenula, Debaryomyces, Mucor, Torulopsis,Methylobacter, Salmonella, Bacillus, Aerobacter, Streptomyces,Escherichia, and Pseudomonas. E. coli is the preferred host.

Accordingly, the present invention provides a recombinant microorganism,useful for the production of 1,3-propanediol comprising: (a) at leastone gene encoding a polypeptide having glycerol-3-phosphatedehydrogenase activity; (b) at least one gene encoding a polypeptidehaving glycerol-3-phosphatase activity; (c) at least one gene encoding apolypeptide having a dehydratase activity; (d) at least one geneencoding a dehydratase reactivation factor; (e) at least one endogenousgene encoding an non-specific catalytic activity sufficient to convert3-hydroxypropionaldehyde to 1,3-propanediol, wherein no functional dhaTgene encoding a 1,3-propanediol oxidoreductase is present. The preferredembodiment is a recombinant microorganism (preferably E. coli) where nodhaT gene is present. Optionally, the recombinant microorganism maycomprise mutations (e.g., deletion mutations or point mutations) inendogenous genes selected from the group consisting of: (a) a geneencoding a polypeptide having glycerol kinase activity; (b) a geneencoding a polypeptide having glycerol dehydrogenase activity; and (c)gene encoding a polypeptide having triosephosphate isomerase activity.

In another embodiment the invention includes a process for theproduction of 1,3-propanediol comprising: (a) contacting, under suitableconditions, a recombinant E. coli comprising a dha regulon and lacking afunctional dhaT gene encoding a 1,3-propanediol oxidoreductase activitywith at least one carbon source, wherein the carbon source is selectedfrom the group consisting of monosaccharides, oligosaccharides,polysaccharides, and single-carbon substrates; and (b) optionallyrecovering the 1,3-propanediol produced in (a).

The invention also provides a process for the production of1,3-propanediol from a recombinant microorganism comprising: (a)contacting the recombinant microorganism of the present invention withat least one carbon source selected from the group consisting ofmonosaccharides, oligosaccharides, polysaccharides, and single-carbonsubstrates whereby 1,3-propanediol is produced; and (b) optionallyrecovering the 1,3-propanediol produced in (a).

Similarly the invention intends to provide a process for the productionof 1,3-propanediol from a recombinant microorganism comprising:

(a) contacting a recombinant microorganism with at least one carbonsource, said recombinant microorganism comprising:

-   -   (i) at least one gene encoding a polypeptide having a        dehydratase activity;    -   (ii) at least one gene encoding a dehydratase reactivation        factor;    -   (iii) at least one endogenous gene encoding a non-specific        catalytic activity sufficient to convert        3-hydroxypropionaldehyde to 1,3-propanediol; wherein no        functional dhaT gene encoding a 1,3-propanediol oxidoreductase        is present;    -   said carbon source selected from the group consisting of        glycerol and dihydroxyacetone, wherein 1,3-propanediol is        produced and;

(b) optionally recovering the 1,3-propanediol produced in (a).

Yet another aspect of the invention provides for the co-feeding of thecarbon substrate. In this embodiment for the production of1,3-propanediol, the steps are: (a) contacting a recombinant E. coliwith a first source of carbon and with a second source of carbon, saidrecombinant E. coli comprising: (i) at least one exogenous gene encodinga polypeptide having a dehydratase activity; (ii) at least one exogenousgene encoding a dehydratase reactivation factor; (iii) at least oneexogenous gene encoding a non-specific catalytic activity sufficient toconvert 3-hydroxypropionaldehyde to 1,3-propanediol, wherein nofunctional dhaT gene encoding a 1,3-propanediol oxidoreductase activityis present in the recombinant E. coli and wherein said first carbonsource is selected from the group consisting of glycerol anddihydroxyacetone, and said second carbon source is selected from thegroup consisting of monosaccharides, oligosaccharides, polysaccharides,and single-carbon substrates, and (b) the 1,3-propanediol produced in(a) is optionally recovered. The co-feed may be sequential orsimultaneous. The recombinant E. coli used in a co-feeding embodimentmay further comprise: (a) a set of exogenous genes consisting of (i) atleast one gene encoding a polypeptide having glycerol-3-phosphatedehydrogenase activity; (ii) at least one gene encoding a polypeptidehaving glycerol-3-phosphatase activity; and (iii) at least one subset ofgenes encoding the gene products of dhaR, orfY, orfX, orfW, dhaB1,dhaB2, dhaB3 and orfZ, and (b) a set of endogenous genes, each genehaving a mutation inactivating the gene, the set consisting of: (i) agene encoding a polypeptide having glycerol kinase activity; (ii) a geneencoding a polypeptide having glycerol dehydrogenase activity; and (iii)a gene encoding a polypeptide having triosephosphate isomerase activity.

Useful recombinant E. coli strains include recombinant E. coli strainKLP23 comprising: (a) a set of two endogenous genes, each gene having amutation inactivating the gene, the set consisting of: (i) a geneencoding a polypeptide having a glycerol kinase activity; and (ii) agene encoding a polypeptide having a glycerol dehydrogenase activity;(b) at least one exogenous gene encoding a polypeptide havingglycerol-3-phosphate dehydrogenase activity; (c) at least one exogenousgene encoding a polypeptide having glycerol-3-phosphatase activity; and(d) a plasmid pKP32 and a recombinant E. coli strain RJ8 comprising: (a)set of three endogenous genes, each gene having a mutation inactivatingthe gene, the set consisting of: (i) a gene encoding a polypeptidehaving a glycerol kinase activity; (ii) a gene encoding a polypeptidehaving a glycerol dehydrogenase activity; and (iii) a gene encoding apolypeptide having a triosephosphate isomerase activity.

Other useful embodiments include recombinant E. coli comprising: (a) aset of exogenous genes consisting of: (i) at least one gene encoding apolypeptide having a dehydratase activity; (ii) at least one geneencoding a polypeptide having glycerol-3-phosphate dehydrogenaseactivity; (iii) at least one gene encoding a polypeptide havingglycerol-3-phosphatase activity; and (iv) at least one gene encoding adehydratase reactivation factor; and (b) at least one endogenous geneencoding a non-specific catalytic activity to convert3-hydroxypropionaldehyde to 1,3-propanediol; wherein no functional dhaTgene encoding a 1,3-propanediol oxidoreductase activity is present inthe recombinant E. coli.

Another embodiment is a recombinant E. coli comprising: (a) a set ofexogenous genes consisting of (i) at least one gene encoding apolypeptide having glycerol-3-phosphate dehydrogenase activity; (ii) atleast one gene encoding a polypeptide having glycerol-3-phosphataseactivity; and (iii) at least one subset of genes encoding the geneproducts of dhaR, orfY, orfX, orfW, dhaB1, dhaB2, dhaB3 and orfZ, and(b) at least one endogenous gene encoding a non-specific catalyticactivity to convert 3-hydroxypropionaldehyde to 1,3-propanediol, whereinno functional dhaT gene encoding a 1,3-propanediol oxidoreductaseactivity is present in the recombinant E. coli. This embodiment alsoincludes a process using a recombinant E. coli further comprising a setof endogenous genes, each gene having a mutation inactivating the gene,the set consisting of: (a) a gene encoding a polypeptide having glycerolkinase activity; (b) a gene encoding a polypeptide having glyceroldehydrogenase activity; and (c) a gene encoding a polypeptide havingtriosephosphate isomerase activity.

This embodiment still further includes a process for the bioproductionof 1,3-propanediol comprising: (a) contacting under suitable conditionsthe immediately disclosed recombinant E. coli with at least one carbonsource selected from the group consisting of monosaccharides,oligosaccharides, polysaccharides, and single-carbon substrates whereby1,3-propanediol is produced; and (b) optionally recovering the1,3-propanediol produced in (a).

And also includes a further process for the bioproduction of1,3-propanediol comprising: (a) contacting the recombinant E. coli ofthe immediately disclosed embodiments that further comprise: (i) atleast one exogenous gene encoding a polypeptide having a dehydrataseactivity; (ii) at least one exogenous gene encoding a dehydratasereactivation factor; (iii) at least one endogenous gene encoding anon-specific catalytic activity to convert 3-hydroxy-propionaldehyde to1,3-propanediol, with at least one carbon source selected from the groupconsisting of glycerol and dihydroxyacetone, and (b) optionallyrecovering the 1,3-propanediol produced in (a).

BRIEF DESCRIPTION OF THE DRAWINGS, SEQUENCE DESCRIPTIONS, AND BIOLOGICALDEPOSITS

The invention can be more fully understood from the following detaileddescription, Figures, the accompanying sequence descriptions, andbiological deposits that form parts of this application.

FIG. 1 presents the gene organization within the sequence of the dharegulon subclone pHK28-26.

FIG. 2 presents a graph of the extracellular soluble protein (g/L)compared between two fermentations runs essentially as described inExample 7 using a constant feed of vitamin B₁₂. In one case, solidlines, the strain used was KLP23/pAH48/pKP32. In the other case, dashedlines, the strain used was KLP23/pAH48/pDT29.

FIG. 3 presents a graph of the cell viability [(viable cells/mL)/OD550]compared between two fermentations runs essentially as described inExample 7 using a constant feed of vitamin B₁₂. In one case (solidlines), the strain used was KLP23/pAH48/pKP32. In the other case (dashedlines), the strain used was KLP23/pAH48/pDT29.

FIG. 4 presents a graph of the yield of glycerol from glucose comparedbetween two fermentations runs essentially as described in Example 7,but in the absence of vitamin B₁₂ or coenzyme B₁₂. In one case (solidlines), the strain used was KLP23/pAH48/pKP32. In the other case (dashedlines), the strain used was KLP23/pAH48/pDT29.

FIG. 5 is a flow diagram illustrating the metabolic conversion ofglucose to 1,3-propanediol.

FIG. 6 is a 2D-PAGE membrane blot with the soluble protein fractionextracted from a band showing endogenous E. coli oxidoreductase activity(non-specific catalytic activity) on a native gel.

The 68 sequence descriptions and the sequence listing attached heretowill comply with the rules governing nucleotide and/or amino acidsequence disclosures in patent applications as set forth in 37 C.F.R.§1.821-1.825 (“Requirements for Patent Applications ContainingNucleotide Sequences and/or Amino Acid Sequence Disclosures—the SequenceRules”) and will be consistent with World Intellectual PropertyOrganization (WIPO) Standard ST2.5 (1998) and the sequence listingrequirements of the EPO and PCT (Rules 5.2 and 49.5(a-bis), and Section208 and Annex C of the Administration Instructions). The SequenceDescriptions contain the one letter code for nucleotide sequencecharacters and the three letter codes for amino acids as defined inconformity with the IUPAC-IYUB standards described in Nucleic Acids Res.13, 3021-3030 (1985) and in the Biochemical Journal 219, 345-373 (1984)which are herein incorporated by reference.

SEQ ID NO:1 contains the nucleotide sequence determined from a 12.1 kbEcoRI-SalI fragment from pKP1 (cosmid containing DNA from Klebsiellapneumoniae), subcloned into pIBI31 (IBI Biosystem, New Haven, Conn.),and termed pHK28-26. Table 1 further details genes, corresponding basepairs identified within SEQ ID NO:1, and associated functionality. Seealso Example 1.

SEQ ID NO:57 contains the amino acid sequence determined for YqhD.

SEQ ID NO:58 contains the nucleotide sequence determined for yqhD.

Applicants have made the following biological deposits under the termsof the Budapest Treaty on the International Recognition of the Depositof Micro-organisms for the Purposes of Patent Procedure:

Int'l Depository Date of Depositor Identification Reference DesignationDeposit E. coli DH5α; transformed ATCC 69789 18 Apr. 1995 with plasmidpKP1comprising a portion of the Klebsiella genome encoding the glyceroldehydratase enzyme E. coli DH5α transformed with ATCC 69790 18 Apr. 1995pKP4 comprising a portion of Klebsiella genome encoding a dioldehydratase enzyme E. coli MSP33.6 comprising a ATCC 98598 25 Nov. 1997Deletion in gldA E. coli RJF10m ATCC 98597 25 Nov. 1997 comprising adeletion in glpK

The deposit(s) will be maintained in the indicated internationaldepository for at least 30 years and will be made available to thepublic upon the grant of a patent disclosing it. The availability of adeposit does not constitute a license to practice the subject inventionin derogation of patent rights granted by government action.

As used herein, “ATCC” refers to the American Type Culture Collectioninternational depository located 10801 University Blvd., Manassas, Va.20110-2209 U.S.A. The “ATCC No.” is the accession number to cultures ondeposit with the ATCC.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides for an improved process for bioconvertinga fermentable carbon source directly to 1,3-propanediol using a singlemicroorganism. The method is characterized by improved titer, yield, andcell viability as well as a decrease in cell lysis during fermentation.

The present invention is based, in part, upon the observation that1,3-propanediol fermentation processes comprising 1,3-propanedioloxidoreductase (dhaT) are characterized by high levels of 3HPA and otheraldehydes and ketones in the medium, which is correlated to a decreasein cell viability. The present invention is also based, in part, uponthe unexpected finding that the model host, E. coli, is capable ofconverting 3-HPA to 1,3-propanediol by an endogenous non-specificcatalytic activity capable of converting 3-hydroxypropionaldehyde to1,3-propanediol. The present invention is further based, in part, uponthe unexpected finding that an E. coli fermentation process comprisingthis non-specific catalytic activity and lacking a functional dhaTresults in increased cell viability during fermentation and provides forhigher titers and/or yields of 1,3-propanediol than a fermentationprocess comprising a functional dhaT.

In one aspect, glycerol is a model substrate, the host microorganism hasa mutation in wild-type dhaT such that there is no 1,3-propanedioloxidoreductase activity and comprises a non-specific catalytic activitysufficient to convert 3-hydroxypropionaldehyde to 1,3-propanediol. Inanother aspect, glucose is a model substrate and recombinant E. coli isa model host. In this aspect, E. coli comprises an endogenousnon-specific catalytic activity sufficient to convert3-hydroxypropionaldehyde to 1,3-propanediol. In one embodiment, thenon-specific catalytic activity is an alcohol dehydrogenase.

In one aspect, the present invention provides a recombinant E. coliexpressing a group of genes comprising (a) at least one gene encoding apolypeptide having glycerol-3-phosphate dehydrogenase activity; (b) atleast one gene encoding a polypeptide having glycerol-3-phosphataseactivity; (c) at least one gene encoding a polypeptide having adehydratase activity; (d) at least one gene encoding a dehydratasereactivation factor; and (e) at least one endogenous gene encoding annon-specific catalytic activity sufficient to convert3-hydroxy-propionaldehyde to 1,3-propanediol; use of this microorganismconverts glucose to 1,3-propanediol at a high titer. In another aspectof this invention, the elimination of the functional dhaT gene in thisrecombinant E. coli provides an unexpectedly higher titer of1,3-propanediol from glucose than previously attained.

The present invention provides an improved method for the biologicalproduction of 1,3-propanediol from a fermentable carbon source in asingle microorganism. In one aspect of the present invention, animproved process for the conversion of glucose to 1,3-propanediol isachieved by the use of a recombinant microorganism comprising a host E.coli transformed with the Klebsiella pneumoniae dha regulon genes dhaR,orfY, dhaT, orfX, orfW, dhaB1, dhaB2, dhaB3, and orfZ, all these genesarranged in the same genetic organization as found in wild typeKlebsiella pneumoniae. The titer obtained for the fermentation processis significantly higher than any titer previously reported for a similarfermentation. This improvement relies on the use of the plasmid pDT29 asdescribed in Example 6 and Example 7.

In another aspect of the present invention, a further improved processfor the production of 1,3-propanediol from glucose is achieved using arecombinant E. coli containing genes encoding a G3PDH, a G3Pphosphatase, a dehydratase, and a dehydratase reactivation factorcompared to a process using a recombinant E. coli containing genesencoding a G3PDH, a G3P phosphatase, a dehydratase, a dehydratasereactivation factor, and also a functional dhaT. The dramaticallyimproved process relies on an endogenous gene encoding a non-specificcatalytic activity, expected to be an alcohol dehydrogenase, which ispresent in E. coli.

The dramatic improvement in the process is evident as an increase in1,3-propanediol titer as illustrated in Examples 7 and 9. Theimprovement in the process is also evident as a decrease in cell lysisas determined by the extracellular soluble protein concentration in thefermentation broth. This aspect of the invention is illustrated in FIG.2. Additionally, the improvement in the process is evident as prolongedcell viability over the course of the fermentation. This aspect of theinvention is illustrated in FIG. 3. Furthermore, the improvement in theprocess is also evident as an increase in yield. In E. coli expressing a1,3-propanediol oxidoreductase (dhaT) (for example, E. coli KLP23transformed with the plasmid pDT29), glycerol can be metabolized to aproduct other than 3-HPA. In direct contrast, in E. coli not expressinga 1,3-propanediol oxidoreductase (dhaT) (for example, E. coli KLP23transformed with the plasmid pKP32), glycerol is not metabolized to aproduct other than 3-HPA. That this cryptic pathway is attributable tothe presence or absence of a functional dhaT is demonstrated by thelower yield of glycerol from glucose as illustrated in FIG. 4.

As used herein the following terms may be used for interpretation of theclaims and specification.

The terms “glycerol-3-phosphate dehydrogenase” and “G3PDH” refer to apolypeptide responsible for an enzyme activity that catalyzes theconversion of dihydroxyacetone phosphate (DHAP) to glycerol-3-phosphate(G3P). In vivo G3PDH may be NADH; NADPH; or FAD-dependent. Whenspecifically referring to a cofactor specific glycerol-3-phosphatedehydrogenase, the terms “NADH-dependent glycerol-3-phosphatedehydrogenase”, “NADPH-dependent glycerol-3-phosphate dehydrogenase” and“FAD-dependent glycerol-3-phosphate dehydrogenase” will be used. As itis generally the case that NADH-dependent and NADPH-dependentglycerol-3-phosphate dehydrogenases are able to use NADH and NADPHinterchangeably (for example by the gene encoded by gpsA), the termsNADH-dependent and NADPH-dependent glycerol-3-phosphate dehydrogenasewill be used interchangeably. The NADH-dependent enzyme (EC 1.1.1.8) isencoded, for example, by several genes including GPD1 (GenBankZ74071×2), or GPD2 (GenBank Z35169×1), or GPD3 (GenBank G984182), orDAR1 (GenBank Z74071×2). The NADPH-dependent enzyme (EC 1.1.1.94) isencoded by gpsA (GenBank U321643, (cds 197911-196892) G466746 andL45246). The FAD-dependent enzyme (EC 1.1.99.5) is encoded by GUT2(GenBank Z47047×23), or glpD (GenBank G147838), or glpABC (GenBankM20938) (see WO 9928480 and references therein, which are hereinincorporated by reference).

The terms “glycerol-3-phosphatase”, “sn-glycerol-3-phosphatase”, or“d,1-glycerol phosphatase”, and “G3P phosphatase” refer to a polypeptideresponsible for an enzyme activity that catalyzes the conversion ofglycerol-3-phosphate and water to glycerol and inorganic phosphate. G3Pphosphatase is encoded, for example, by GPP1 (GenBank Z47047×125), orGPP2 (GenBank U18813×11) (see WO 9928480 and references therein, whichare herein incorporated by reference).

The term “glycerol kinase” refers to a polypeptide responsible for anenzyme activity that catalyzes the conversion of glycerol and ATP toglycerol-3-phosphate and ADP. The high-energy phosphate donor ATP may bereplaced by physiological substitutes (e.g., phosphoenolpyruvate).Glycerol kinase is encoded, for example, by GUT1 (GenBank U11583×19) andglpK (GenBank L19201) (see WO 9928480 and references therein, which areherein incorporated by reference).

The term “glycerol dehydrogenase” refers to a polypeptide responsiblefor an enzyme activity that catalyzes the conversion of glycerol todihydroxyacetone (E.C. 1.1.1.6) or glycerol to glyceraldehyde (E.C.1.1.1.72). A polypeptide responsible for an enzyme activity thatcatalyzes the conversion of glycerol to dihydroxyacetone is alsoreferred to as a “dihydroxyacetone reductase”. Glycerol dehydrogenasemay be dependent upon NADH (E.C. 1.1.1.6), NADPH (E.C. 1.1.1.72), orother cofactors (e.g., E.C. 1.1.99.22). A NADH-dependent glyceroldehydrogenase is encoded, for example, by gldA (GenBank U00006) (see WO9928480 and references therein, which are herein incorporated byreference).

The term “dehydratase enzyme” or “dehydratase” will refer to any enzymeactivity that catalyzes the conversion of a glycerol molecule to theproduct 3-hydroxypropionaldehyde. For the purposes of the presentinvention the dehydratase enzymes include a glycerol dehydratase (E.C.4.2.1.30) and a diol dehydratase (E.C. 4.2.1.28) having preferredsubstrates of glycerol and 1,2-propanediol, respectively. Genes fordehydratase enzymes have been identified in Klebsiella pneumoniae,Citrobacter freundii, Clostridium pasteurianum, Salmonella typhimurium,and Klebsiella oxytoca. In each case, the dehydratase is composed ofthree subunits: the large or “α” subunit, the medium or “β” subunit, andthe small or “γ” subunit. Due to the wide variation in gene nomenclatureused in the literature, a comparative chart is given in Table 1 tofacilitate identification. The genes are also described in, for example,Daniel et al. (FEMS Microbiol. Rev. 22, 553 (1999)) and Toraya and Mori(J. Biol. Chem. 274, 3372 (1999)). Referring to Table 1, genes encodingthe large or “α” subunit of glycerol dehydratase include dhaB1, gldA anddhaB; genes encoding the medium or “β” subunit include dhaB2, gldB, anddhaC; genes encoding the small or “γ” subunit include dhaB3, gldC, anddhaE. Also referring to Table 1, genes encoding the large or “α” subunitof diol dehydratase include pduC and pddA; genes encoding the medium or“β” subunit include pduD and pddB; genes encoding the small or “γ”subunit include pduE and pddC.

TABLE 1 Comparative chart of gene names and GenBank references fordehydratase and dehydratase linked functions. GENE FUNCTION: 1,3-PDregulatory unknown reactivation dehydrogenase unknown ORGANISM (GenBankReference) gene base pairs gene base pairs geme base pairs gene basepairs gene base pairs K. pneumoniae (SEQ ID NO: 1) dhaR 2209-4134 orfW4112-4642 orfX 4643-4996 dhaT 5017-6108 orfY 6202-6630 K. pneumoniae(U30903) orf2c 7116-7646 orf2b 6762-7115 dhaT 5578-6741 orf2a 5125-5556K. pneumoniae (U60992) gdrB C. freundii (U09771) dhaR 3746-5671 orfW5649-6179 orfX 6180-6533 dhaT 6550-7713 orfY 7736-8164 C. pasteurianum(AF051373) C. pasteurianum (AF006034) orfW 210-731 orfX  1-196 dhaT1232-2389 orfY  746-1177 S. typhimurium (AF026270) pduH 8274-8645 K.oxytoca (AF017781) ddrB 2063-2440 K. oxytoca (AF051373) GENE FUNCTION:dehydratase, α dehydratase, β dehydratase, γ reactivation ORGANISM(GenBank Reference) gene base pairs gene base pairs gene base pairs genebase pairs K. pneumoniae (SEQ ID NO: 1) dhaB1 7044-8711 dhaB2 8724-9308dhaB3 9311-9736 orfZ  9749-11572 K. pneumoniae (U30903) dhaB1 3047-4714dhaB2 2450-2890 dhaB3 2022-2447 dhaB4  186-2009 K. pneumoniae (U60992)gldA  121-1788 gldB 1801-2385 gldC 2388-2813 gdrA C. freundii (U09771)dhaB  8556-10223 dhaC 10235-10819 dhaE 10822-11250 orfZ 11261-13072 C.pasteurianum (AF051373) dhaB  84-1748 dhaC 1779-2318 dhaE 2333-2773 orfZ2790-4598 C. pasteurianum (AF006034) S. typhimurium (AF026270) pduC3557-5221 pduD 5232-5906 pduE 5921-6442 pduG 6452-8284 K. oxytoca(AF017781) ddrA  241-2073 K. oxytoca (AF051373) pddA  121-1785 pddB1796-2470 pddC 2485-3006

Glycerol and diol dehydratases are subject to mechanism-based suicideinactivation by glycerol and some other substrates (Daniel et al., FEMSMicrobiol. Rev. 22, 553 (1999)). The term “dehydratase reactivationfactor” refers to those proteins responsible for reactivating thedehydratase activity. The terms “dehydratase reactivating activity”,“reactivating the dehydratase activity” or “regenerating the dehydrataseactivity” refers to the phenomenon of converting a dehydratase notcapable of catalysis of a substrate to one capable of catalysis of asubstrate or to the phenomenon of inhibiting the inactivation of adehydratase or the phenomenon of extending the useful half-life of thedehydratase enzyme in vivo. Two proteins have been identified as beinginvolved as the dehydratase reactivation factor (see WO 9821341 (U.S.Pat. No. 6,013,494) and references therein, which are hereinincorporated by reference; Daniel et al., supra; Toraya and Mori, J.Biol. Chem. 274, 3372 (1999); and Tobimatsu et al., J. Bacteriol. 181,4110 (1999)). Referring to Table 1, genes encoding one of the proteinsinclude orfZ, dhaB4, gdrA, pduG and ddrA. Also referring to Table 1,genes encoding the second of the two proteins include orfX, orf2b, gdrB,pduH and ddrB.

The terms “1,3-propanediol oxidoreductase”, “1,3-propanedioldehydrogenase” or “DhaT” refer to the polypeptide(s) responsible for anenzyme activity that is capable of catalyzing the interconversion of3-HPA and 1,3-propanediol provided the gene(s) encoding such activity isfound to be physically or transcriptionally linked to a dehydrataseenzyme in its natural (i.e., wild type) setting; for example, the geneis found within a dha regulon as is the case with dhaT from Klebsiellapneumonia. Referring to Table 1, genes encoding a 1,3-propanedioloxidoreductase include dhaT from Klebsiella pneumoniae, Citrobacterfreundii, and Clostridium pasteurianum. Each of these genes encode apolypeptide belonging to the family of type III alcohol dehydrogenases,exhibits a conserved iron-binding motif, and has a preference for theNAD⁺/NADH linked interconversion of 3-HPA and 1,3-propanediol (Johnsonand Lin, J. Bacteriol. 169, 2050 (1987); Daniel et al., J. Bacteriol.177, 2151 (1995); and Leurs et al., FEMS Microbiol. Lett. 154, 337(1997)). Enzymes with similar physical properties have been isolatedfrom Lactobacillus brevis and Lactobacillus buchneri (Veiga da Dunha andFoster, Appl. Environ. Microbiol. 58, 2005 (1992)).

The term “dha regulon” refers to a set of associated genes or openreading frames encoding various biological activities, including but notlimited to a dehydratase activity, a reactivation activity, and a1,3-propanediol oxidoreductase. Typically a dha regulon comprises theopen reading frames dhaR, orfY, dhaT, orfX, orfW, dhaB1, dhaB2, dhaB3,and orfZ as described herein.

The term “non-specific catalytic activity” refers to the polypeptide(s)responsible for an enzyme activity that is sufficient to catalyze theinterconversion of 3-HPA and 1,3-propanediol and specifically excludes1,3-propanediol oxidoreductase(s). Typically these enzymes are alcoholdehydrogenases. Such enzymes may utilize cofactors other than NAD⁺/NADH,including but not limited to flavins such as FAD or FMN. A gene(s) for anon-specific alcohol dehydrogenase(s) is found, for example, to beendogenously encoded and functionally expressed within the microorganismE. coli KLP23.

The terms “function” or “enzyme function” refer to the catalyticactivity of an enzyme in altering the energy required to perform aspecific chemical reaction. It is understood that such an activity mayapply to a reaction in equilibrium where the production of eitherproduct or substrate may be accomplished under suitable conditions.

The terms “polypeptide” and “protein” are used interchangeably.

The terms “carbon substrate” and “carbon source” refer to a carbonsource capable of being metabolized by host microorganisms of thepresent invention and particularly carbon sources selected from thegroup consisting of monosaccharides, oligosaccharides, polysaccharides,and one-carbon substrates or mixtures thereof.

The terms “host cell” or “host microorganism” refer to a microorganismcapable of receiving foreign or heterologous genes and of expressingthose genes to produce an active gene product.

The terms “foreign gene”, “foreign DNA”, “heterologous gene” and“heterologous DNA” refer to genetic material native to one organism thathas been placed within a host microorganism by various means. The geneof interest may be a naturally occurring gene, a mutated gene, or asynthetic gene.

The terms “transformation” and “transfection” refer to the acquisitionof new genes in a cell after the incorporation of nucleic acid. Theacquired genes may be integrated into chromosomal DNA or introduced asextrachromosomal replicating sequences. The term “transformant” refersto the product of a transformation.

The term “genetically altered” refers to the process of changinghereditary material by transformation or mutation.

The terms “recombinant microorganism” and “transformed host” refer toany microorganism having been transformed with heterologous or foreigngenes or extra copies of homologous genes. The recombinantmicroorganisms of the present invention express foreign genes encodingglycerol-3-phosphate dehydrogenase (GPD1), glycerol-3-phosphatase(GPP2), glycerol dehydratase (dhaB1, dhaB2 and dhaB3), dehydratasereactivation factor (orfZ and orfX), and optionally 1,3-propanedioloxidoreductase (dhaT) for the production of 1,3-propanediol fromsuitable carbon substrates. A preferred embodiment is an E. colitransformed with these genes but lacking a functional dhaT. A hostmicroorganism, other than E. coli, may also be transformed to containthe disclosed genes and the gene for the non-specific catalytic activityfor the interconversion of 3-HPA and 1,3-propanediol, specificallyexcluding 1,3-propanediol oxidoreductase(s) (dhaT).

“Gene” refers to a nucleic acid fragment that expresses a specificprotein, including regulatory sequences preceding (5′ non-coding) andfollowing (3′ non-coding) the coding region. The terms “native” and“wild-type” refer to a gene as found in nature with its own regulatorysequences.

The terms “encoding” and “coding” refer to the process by which a gene,through the mechanisms of transcription and translation, produces anamino acid sequence. It is understood that the process of encoding aspecific amino acid sequence includes DNA sequences that may involvebase changes that do not cause a change in the encoded amino acid, orwhich involve base changes which may alter one or more amino acids, butdo not affect the functional properties of the protein encoded by theDNA sequence. It is therefore understood that the invention encompassesmore than the specific exemplary sequences.

The term “isolated” refers to a protein or DNA sequence that is removedfrom at least one component with which it is naturally associated.

An “isolated nucleic acid molecule” is a polymer of RNA or DNA that issingle- or double-stranded, optionally containing synthetic, non-naturalor altered nucleotide bases. An isolated nucleic acid molecule in theform of a polymer of DNA may be comprised of one or more segments ofcDNA, genomic DNA or synthetic DNA.

“Substantially similar” refers to nucleic acid molecules wherein changesin one or more nucleotide bases result in substitution of one or moreamino acids, but do not affect the functional properties of the proteinencoded by the DNA sequence. “Substantially similar” also refers tonucleic acid molecules wherein changes in one or more nucleotide basesdo not affect the ability of the nucleic acid molecule to mediatealteration of gene expression by antisense or co-suppression technology.“Substantially similar” also refers to modifications of the nucleic acidmolecules of the instant invention (such as deletion or insertion of oneor more nucleotide bases) that do not substantially affect thefunctional properties of the resulting transcript vis-à-vis the abilityto mediate alteration of gene expression by antisense or co-suppressiontechnology or alteration of the functional properties of the resultingprotein molecule. The invention encompasses more than the specificexemplary sequences.

For example, it is well known in the art that alterations in a genewhich result in the production of a chemically equivalent amino acid ata given site, but do not effect the functional properties of the encodedprotein are common. For the purposes of the present inventionsubstitutions are defined as exchanges within one of the following fivegroups:

-   -   1. Small aliphatic, nonpolar or slightly polar residues: Ala,        Ser, Thr (Pro, Gly);    -   2. Polar, negatively charged residues and their amides: Asp,        Asn, Glu, Gln;    -   3. Polar, positively charged residues: His, Arg, Lys;    -   4. Large aliphatic, nonpolar residues: Met, Leu, Ile, Val (Cys);        and    -   5. Large aromatic residues: Phe, Tyr, Trp.

Thus, a codon for the amino acid alanine, a hydrophobic amino acid, maybe substituted by a codon encoding another less hydrophobic residue(such as glycine) or a more hydrophobic residue (such as valine,leucine, or isoleucine). Similarly, changes which result in substitutionof one negatively charged residue for another (such as aspartic acid forglutamic acid) or one positively charged residue for another (such aslysine for arginine) can also be expected to produce a functionallyequivalent product.

In many cases, nucleotide changes which result in alteration of theN-terminal and C-terminal portions of the protein molecule would alsonot be expected to alter the activity of the protein.

Each of the proposed modifications is well within the routine skill inthe art, as is determination of retention of biological activity of theencoded products. Moreover, the skilled artisan recognizes thatsubstantially similar sequences encompassed by this invention are alsodefined by their ability to hybridize, under stringent conditions(0.1×SSC, 0.1% SDS, 65° C. and washed with 2×SSC, 0.1% SDS followed by0.1×SSC, 0.1% SDS), with the sequences exemplified herein. Preferredsubstantially similar nucleic acid fragments of the instant inventionare those nucleic acid fragments whose DNA sequences are at least 80%identical to the DNA sequence of the nucleic acid fragments reportedherein. More preferred nucleic acid fragments are at least 90% identicalto the DNA sequence of the nucleic acid fragments reported herein. Mostpreferred are nucleic acid fragments that are at least 95% identical tothe DNA sequence of the nucleic acid fragments reported herein.

A nucleic acid fragment is “hybridizable” to another nucleic acidfragment, such as a cDNA, genomic DNA, or RNA, when a single strandedform of the nucleic acid fragment can anneal to the other nucleic acidfragment under the appropriate conditions of temperature and solutionionic strength. Hybridization and washing conditions are well known andexemplified in Sambrook, J., Fritsch, E. F. and Maniatis, T. MolecularCloning: A Laboratory Manual, Second Edition, Cold Spring HarborLaboratory Press, Cold Spring Harbor (1989), particularly Chapter 11 andTable 11.1 therein (entirely incorporated herein by reference). Theconditions of temperature and ionic strength determine the “stringency”of the hybridization. For preliminary screening for homologous nucleicacids, low stringency hybridization conditions, corresponding to a Tm of55°, can be used, e.g., 5×SSC, 0.1% SDS, 0.25% milk, and no formamide;or 30% formamide, 5×SSC, 0.5% SDS. Moderate stringency hybridizationconditions correspond to a higher Tm, e.g., 40% formamide, with 5× or6×SSC. Hybridization requires that the two nucleic acids containcomplementary sequences, although depending on the stringency of thehybridization, mismatches between bases are possible. The appropriatestringency for hybridizing nucleic acids depends on the length of thenucleic acids and the degree of complementation, variables well known inthe art. The greater the degree of similarity or homology between twonucleotide sequences, the greater the value of Tm for hybrids of nucleicacids having those sequences. The relative stability (corresponding tohigher Tm) of nucleic acid hybridization decreases in the followingorder: RNA:RNA, DNA:RNA, DNA:DNA. For hybrids of greater than 100nucleotides in length, equations for calculating Tm have been derived(see Sambrook et al., supra, 9.50-9.51). For hybridization with shorternucleic acids, i.e., oligonucleotides, the position of mismatchesbecomes more important, and the length of the oligonucleotide determinesits specificity (see Sambrook et al., supra, 11.7-11.8). In oneembodiment the length for a hybridizable nucleic acid is at least about10 nucleotides. Preferable a minimum length for a hybridizable nucleicacid is at least about 15 nucleotides; more preferably at least about 20nucleotides; and most preferably the length is at least 30 nucleotides.Furthermore, the skilled artisan will recognize that the temperature andwash solution salt concentration may be adjusted as necessary accordingto factors such as length of the probe.

A “substantial portion” refers to an amino acid or nucleotide sequencewhich comprises enough of the amino acid sequence of a polypeptide orthe nucleotide sequence of a gene to afford putative identification ofthat polypeptide or gene, either by manual evaluation of the sequence byone skilled in the art, or by computer-automated sequence comparison andidentification using algorithms such as BLAST (Basic Local AlignmentSearch Tool; Altschul et al., J. Mol. Biol. 215:403-410 (1993); see alsowww.ncbi.nlm.nih.gov/BLAST/). In general, a sequence of ten or morecontiguous amino acids or thirty or more nucleotides is necessary inorder to putatively identify a polypeptide or nucleic acid sequence ashomologous to a known protein or gene. Moreover, with respect tonucleotide sequences, gene-specific oligonucleotide probes comprising20-30 contiguous nucleotides may be used in sequence-dependent methodsof gene identification (e.g., Southern hybridization) and isolation(e.g., in situ hybridization of bacterial colonies or bacteriophageplaques). In addition, short oligonucleotides of 12-15 bases may be usedas amplification primers in PCR in order to obtain a particular nucleicacid molecule comprising the primers. Accordingly, a “substantialportion” of a nucleotide sequence comprises enough of the sequence toafford specific identification and/or isolation of a nucleic acidmolecule comprising the sequence. The instant specification teachespartial or complete amino acid and nucleotide sequences encoding one ormore particular proteins. The skilled artisan, having the benefit of thesequences as reported herein, may now use all or a substantial portionof the disclosed sequences for the purpose known to those skilled in theart. Accordingly, the instant invention comprises the complete sequencesas reported in the accompanying Sequence Listing, as well as substantialportions of those sequences as defined above.

The term “complementary” describes the relationship between nucleotidebases that are capable to hybridizing to one another. For example, withrespect to DNA, adenosine is complementary to thymine and cytosine iscomplementary to guanine. Accordingly, the instant invention alsoincludes isolated nucleic acid molecules that are complementary to thecomplete sequences as reported in the accompanying Sequence Listing aswell as those substantially similar nucleic acid sequences.

The term “percent identity”, as known in the art, is a relationshipbetween two or more polypeptide sequences or two or more polynucleotidesequences, as determined by comparing the sequences. In the art,“identity” also means the degree of sequence relatedness betweenpolypeptide or polynucleotide sequences, as the case may be, asdetermined by the match between strings of such sequences. “Identity”and “similarity” can be readily calculated by known methods, includingbut not limited to those described in: Computational Molecular Biology;Lesk, A. M., Ed.; Oxford University Press: New York, 1988; Biocomputing:Informatics and Genome Projects; Smith, D. W., Ed.; Academic Press: NewYork, 1993; Computer Analysis of Sequence Data, Part I; Griffin, A. M.and Griffin, H. G., Eds.; Humana Press: New Jersey, 1994; SequenceAnalysis in Molecular Biology; von Heinje, G., Ed.; Academic Press: NewYork, 1987; and Sequence Analysis Primer; Gribskov, M. and Devereux, J.,Eds.; Stockton Press: New York, 1991. Preferred methods to determineidentity are designed to give the largest match between the sequencestested.

Methods to determine identity and similarity are codified in publiclyavailable computer programs. Preferred computer program methods todetermine identity and similarity between two sequences include, but arenot limited to, the GCG Pileup program found in the GCG program package,using the Needleman and Wunsch algorithm with their standard defaultvalues of gap creation penalty=12 and gap extension penalty=4 (Devereuxet al., Nucleic Acids Res. 12:387-395 (1984)), BLASTP, BLASTN, and FASTA(Pearson et al., Proc. Natl. Acad. Sci. USA 85:2444-2448 (1988). TheBLASTX program is publicly available from NCBI and other sources (BLASTManual, Altschul et al., Natl. Cent. Biotechnol. Inf., Natl. LibraryMed. (NCBI NLM) NIH, Bethesda, Md. 20894; Altschul et al., J. Mol. Biol.215:403-410 (1990); Altschul et al., “Gapped BLAST and PSI-BLAST: a newgeneration of protein database search programs”, Nucleic Acids Res.25:3389-3402 (1997)). Another preferred method to determine percentidentity, is by the method of DNASTAR protein alignment protocol usingthe Jotun-Hein algorithm (Hein et al., Methods Enzymol. 183:626-645(1990)). Default parameters for the Jotun-Hein method for alignmentsare: for multiple alignments, gap penalty=11, gap length penalty=3; forpairwise alignments ktuple=6. As an illustration, by a polynucleotidehaving a nucleotide sequence having at least, for example, 95%“identity” to a reference nucleotide sequence it is intended that thenucleotide sequence of the polynucleotide is identical to the referencesequence except that the polynucleotide sequence may include up to fivepoint mutations per each 100 nucleotides of the reference nucleotidesequence. In other words, to obtain a polynucleotide having a nucleotidesequence at least 95% identical to a reference nucleotide sequence, upto 5% of the nucleotides in the reference sequence may be deleted orsubstituted with another nucleotide, or a number of nucleotides up to 5%of the total nucleotides in the reference sequence may be inserted intothe reference sequence. These mutations of the reference sequence mayoccur at the 5′ or 3′ terminal positions of the reference nucleotidesequence or anywhere between those terminal positions, interspersedeither individually among nucleotides in the reference sequence or inone or more contiguous groups within the reference sequence.Analogously, by a polypeptide having an amino acid sequence having atleast, for example, 95% identity to a reference amino acid sequence isintended that the amino acid sequence of the polypeptide is identical tothe reference sequence except that the polypeptide sequence may includeup to five amino acid alterations per each 100 amino acids of thereference amino acid. In other words, to obtain a polypeptide having anamino acid sequence at least 95% identical to a reference amino acidsequence, up to 5% of the amino acid residues in the reference sequencemay be deleted or substituted with another amino acid, or a number ofamino acids up to 5% of the total amino acid residues in the referencesequence may be inserted into the reference sequence. These alterationsof the reference sequence may occur at the amino or carboxy terminalpositions of the reference amino acid sequence or anywhere between thoseterminal positions, interspersed either individually among residues inthe reference sequence or in one or more contiguous groups within thereference sequence.

The term “homologous” refers to a protein or polypeptide native ornaturally occurring in a given host cell. The invention includesmicroorganisms producing homologous proteins via recombinant DNAtechnology.

The term “percent homology” refers to the extent of amino acid sequenceidentity between polypeptides. When a first amino acid sequence isidentical to a second amino acid sequence, then the first and secondamino acid sequences exhibit 100% homology. The homology between any twopolypeptides is a direct function of the total number of matching aminoacids at a given position in either sequence, e.g., if half of the totalnumber of amino acids in either of the two sequences are the same thenthe two sequences are said to exhibit 50% homology.

“Codon degeneracy” refers to divergence in the genetic code permittingvariation of the nucleotide sequence without effecting the amino acidsequence of an encoded polypeptide. Accordingly, the instant inventionrelates to any nucleic acid molecule that encodes all or a substantialportion of the amino acid sequence as set forth in SEQ ID NO:57. Theskilled artisan is well aware of the “codon-bias” exhibited by aspecific host cell in usage of nucleotide codons to specify a givenamino acid. Therefore, when synthesizing a gene for improved expressionin a host cell, it is desirable to design the gene such that itsfrequency of codon usage approaches the frequency of preferred codonusage of the host cell.

Modifications to the sequence, such as deletions, insertions, orsubstitutions in the sequence which produce silent changes that do notsubstantially affect the functional properties of the resulting proteinmolecule are also contemplated. For example, alteration in the genesequence which reflect the degeneracy of the genetic code, or whichresult in the production of a chemically equivalent amino acid at agiven site, are contemplated. Thus, a codon for the amino acid alanine,a hydrophobic amino acid, may be substituted by a codon encoding anotherless hydrophobic residue, such as glycine, or a more hydrophobicresidue, such as valine, leucine, or isoleucine. Similarly, changeswhich result in substitution of one negatively charged residue foranother, such as aspartic acid for glutamic acid, or one positivelycharged residue for another, such as lysine for arginine, can also beexpected to produce a biologically equivalent product. Nucleotidechanges which result in alteration of the N-terminal and C-terminalportions of the protein molecule would also not be expected to alter theactivity of the protein. In some cases, it may in fact be desirable tomake mutants of the sequence in order to study the effect of alterationon the biological activity of the protein. Each of the proposedmodifications is well within the routine skill in the art, as isdetermination of retention of biological activity in the encodedproducts. Moreover, the skilled artisan recognizes that sequencesencompassed by this invention are also defined by their ability tohybridize, under stringent conditions (0.1×SSC, 0.1% SDS, 65° C.), withthe sequences exemplified herein.

The term “expression” refers to the transcription and translation togene product from a gene coding for the sequence of the gene product.

The terms “plasmid”, “vector”, and “cassette” refer to an extrachromosomal element often carrying genes which are not part of thecentral metabolism of the cell, and usually in the form of circulardouble-stranded DNA molecules. Such elements may be autonomouslyreplicating sequences, genome integrating sequences, phage or nucleotidesequences, linear or circular, of a single- or double-stranded DNA orRNA, derived from any source, in which a number of nucleotide sequenceshave been joined or recombined into a unique construction which iscapable of introducing a promoter fragment and DNA sequence for aselected gene product along with appropriate 3′ untranslated sequenceinto a cell. “Transformation cassette” refers to a specific vectorcontaining a foreign gene and having elements in addition to the foreigngene that facilitates transformation of a particular host cell.“Expression cassette” refers to a specific vector containing a foreigngene and having elements in addition to the foreign gene that allow forenhanced expression of that gene in a foreign host.

Construction of Recombinant Organisms

Recombinant organisms containing the necessary genes that will encodethe enzymatic pathway for the conversion of a carbon substrate to1,3-propanediol may be constructed using techniques well known in theart. Genes encoding glycerol-3-phosphate dehydrogenase (GPD1),glycerol-3-phosphatase (GPP2), glycerol dehydratase (dhaB1, dhaB2, anddhaB3), dehydratase reactivation factor (orfZ and orfX) and1,3-propanediol oxidoreductase (dhaT) were isolated from a native hostsuch as Klebsiella or Saccharomyces and used to transform host strainssuch as E. coli DH5α, ECL707, AA200, or KLP23.

Isolation of Genes

Methods of obtaining desired genes from a bacterial genome are commonand well known in the art of molecular biology. For example, if thesequence of the gene is known, suitable genomic libraries may be createdby restriction endonuclease digestion and may be screened with probescomplementary to the desired gene sequence. Once the sequence isisolated, the DNA may be amplified using standard primer directedamplification methods such as polymerase chain reaction (PCR) (U.S. Pat.No. 4,683,202) to obtain amounts of DNA suitable for transformationusing appropriate vectors.

Alternatively, cosmid libraries may be created where large segments ofgenomic DNA (35-45 kb) may be packaged into vectors and used totransform appropriate hosts. Cosmid vectors are unique in being able toaccommodate large quantities of DNA. Generally cosmid vectors have atleast one copy of the cos DNA sequence which is needed for packaging andsubsequent circularization of the foreign DNA. In addition to the cossequence these vectors will also contain an origin of replication suchas ColE1 and drug resistance markers such as a gene resistant toampicillin or neomycin. Methods of using cosmid vectors for thetransformation of suitable bacterial hosts are well described inSambrook, J. et al., Molecular Cloning: A Laboratory Manual, SecondEdition (1989) Cold Spring Harbor Laboratory Press, herein incorporatedby reference.

Typically to clone cosmids, foreign DNA is isolated and ligated, usingthe appropriate restriction endonucleases, adjacent to the cos region ofthe cosmid vector. Cosmid vectors containing the linearized foreign DNAare then reacted with a DNA packaging vehicle such as bacteriophage.During the packaging process the cos sites are cleaved and the foreignDNA is packaged into the head portion of the bacterial viral particle.These particles are then used to transfect suitable host cells such asE. coli. Once injected into the cell, the foreign DNA circularizes underthe influence of the cos sticky ends. In this manner large segments offoreign DNA can be introduced and expressed in recombinant host cells.

Isolation and Cloning of Genes Encoding Glycerol Dehydratase (dhaB1,dhaB2, and dhaB3) Dehydratase Reactivating Factors (orfZ and orfX), and1,3-Propanediol Dehydrogenase (dhaT)

Cosmid vectors and cosmid transformation methods were used within thecontext of the present invention to clone large segments of genomic DNAfrom bacterial genera known to possess genes capable of processingglycerol to 1,3-propanediol. Specifically, genomic DNA from K.pneumoniae was isolated by methods well known in the art and digestedwith the restriction enzyme Sau3A for insertion into a cosmid vectorSupercos 1 and packaged using GigapackII packaging extracts. Followingconstruction of the vector E. coli XL1-Blue MR cells were transformedwith the cosmid DNA. Transformants were screened for the ability toconvert glycerol to 1,3-propanediol by growing the cells in the presenceof glycerol and analyzing the media for 1,3-propanediol formation.

Two of the 1,3-propanediol positive transformants were analyzed and thecosmids were named pKP1 and pKP2. DNA sequencing revealed extensivehomology to the glycerol dehydratase gene from C. freundii,demonstrating that these transformants contained DNA encoding theglycerol dehydratase gene. Other 1,3-propanediol positive transformantswere analyzed and the cosmids were named pKP4 and pKP5. DNA sequencingrevealed that these cosmids carried DNA encoding a diol dehydratasegene.

Although the instant invention utilizes the isolated genes from within aKlebsiella cosmid, alternate sources of dehydratase genes anddehydratase reactivation factor genes include, but are not limited to,Citrobacter, Clostridia and Salmonella (see Table 1).

Genes Encoding G3PDH and G3P Phosphatase

The present invention provides genes suitable for the expression ofG3PDH and G3P phosphatase activities in a host cell.

Genes encoding G3PDH are known. For example, GPD1 has been isolated fromSaccharomyces and has the base sequence given by SEQ ID NO:53, encodingthe amino acid sequence given in SEQ ID NO:54 (Wang et al., supra).Similarly, G3PDH activity has also been isolated from Saccharomycesencoded by GPD2 (Eriksson et al., Mol. Microbiol. 17, 95 (1995)).

For the purposes of the present invention it is contemplated that anygene encoding a polypeptide responsible for NADH-dependent G3PDHactivity is suitable wherein that activity is capable of catalyzing theconversion of dihydroxyacetone phosphate (DHAP) to glycerol-3-phosphate(G3P). Further, it is contemplated that any gene encoding the amino acidsequence of NADH-dependent G3PDH's corresponding to the genes DAR1,GPD1, GPD2, GPD3, and gpsA will be functional in the present inventionwherein that amino acid sequence may encompass amino acid substitutions,deletions or additions that do not alter the function of the enzyme. Theskilled person will appreciate that genes encoding G3PDH isolated fromother sources will also be suitable for use in the present invention.Genes encoding G3P phosphatase are known. For example, GPP2 has beenisolated from Saccharomyces cerevisiae and has the base sequence givenby SEQ ID NO:55, which encodes the amino acid sequence given in SEQ IDNO:56 (Norbeck et al., J. Biol. Chem. 271, 13875 (1996)).

For the purposes of the present invention, any gene encoding a G3Pphosphatase activity is suitable for use in the method wherein thatactivity is capable of catalyzing the conversion of glycerol-3-phosphateplus H₂O to glycerol plus inorganic phosphate. Further, any geneencoding the amino acid sequence of G3P phosphatase corresponding to thegenes GPP2 and GPP1 will be functional in the present inventionincluding any amino acid sequence that encompasses amino acidsubstitutions, deletions or additions that do not alter the function ofthe G3P phosphatase enzyme. The skilled person will appreciate thatgenes encoding G3P phosphatase isolated from other sources will also besuitable for use in the present invention.

Host Cells

Suitable host cells for the recombinant production of 1,3-propanediolmay be either prokaryotic or eukaryotic and will be limited only by thehost cell ability to express the active enzymes for the 1,3-propanediolpathway. Suitable host cells will be bacteria such as Citrobacter,Enterobacter, Clostridium, Klebsiella, Aerobacter, Lactobacillus,Aspergillus, Saccharomyces, Schizosaccharomyces, Zygosaccharomyces,Pichia, Kluyveromyces, Candida, Hansenula, Debaryomyces, Mucor,Torulopsis, Methylobacter, Escherichia, Salmonella, Bacillus,Streptomyces, and Pseudomonas. Preferred in the present invention are E.coli, E. blattae, Klebsiella, Citrobacter, and Aerobacter.

Microorganisms can be converted to a high titer 1,3-propanediol producerby using the following general protocol.

-   -   1. Determine the presence in a potential host organism of an        endogenous dhaT-like activity that allows for the steady state        concentration of a toxic or inhibitory level of 3-HPA in the        presence of 1-2 M 1,3-propanediol.    -   2. If such an activity exists in the potential host organism,        perform suitable mutagenisis to delete or inactivate this        activity. Confirmation of a non-functional or deleted dhaT-like        activity can be detected by the lack of 3-HPA accumulation in        the presence of 1-2 M 1,3-propanediol.    -   3. Express appropriate genes for a) glycerol production, if        glycerol is not the carbon source, b) glycerol dehydratase and        the associated maintenance system, and c) yqhD.

Considerations which would need to be taken with respect to certainmicroorganisms concern the expression or repression of endogenousdhaT-like enzymes under the conditions for 1,3-propanediol production.These might also include the presence of glycerol, glucose oranaerobisis.

Vectors and Expression Cassettes

The present invention provides a variety of vectors and transformationand expression cassettes suitable for the cloning, transformation andexpression of G3PDH, G3P phosphatase, dehydratase, and dehydratasereactivation factor into a suitable host cell. Suitable vectors will bethose which are compatible with the microorganism employed. Suitablevectors can be derived, for example, from a bacteria, a virus (such asbacteriophage T7 or a M-13 derived phage), a cosmid, a yeast or a plant.Protocols for obtaining and using such vectors are known to those in theart (Sambrook et al., Molecular Cloning: A Laboratory Manual—volumes 1,2, 3 (Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y., 1989)).

Typically, the vector or cassette contains sequences directingtranscription and translation of the appropriate gene, a selectablemarker, and sequences allowing autonomous replication or chromosomalintegration. Suitable vectors comprise a region 5′ of the gene, whichharbors transcriptional initiation controls, and a region 3′ of the DNAfragment which controls transcriptional termination. It is mostpreferred when both control regions are derived from genes homologous tothe transformed host cell. Such control regions need not be derived fromthe genes native to the specific species chosen as a production host.

Initiation control regions, or promoters, which are useful to driveexpression of the G3PDH and G3P phosphatase genes (DAR1 and GPP2,respectively) in the desired host cell are numerous and familiar tothose skilled in the art. Virtually any promoter capable of drivingthese genes is suitable for the present invention including but notlimited to CYC1, HIS3, GAL1, GAL10, ADH1, PGK, PHO5, GAPDH, ADC1, TRP1,URA3, LEU2, ENO, and TPI (useful for expression in Saccharomyces); AOX1(useful for expression in Pichia); and lac, trp, λP_(L), λP_(R), T7,tac, and trc (useful for expression in E. coli).

Termination control regions may also be derived from various genesnative to the preferred hosts. Optionally, a termination site may beunnecessary; however, it is most preferred if included.

For effective expression of the instant enzymes, DNA encoding theenzymes are linked operably through initiation codons to selectedexpression control regions such that expression results in the formationof the appropriate messenger RNA.

Particularly useful in the present invention are the vectors pDT29 andpKP32 which are designed to be used in conjunction with pAH48. Theessential elements of pDT29 and pKP32 are derived from the dha regulonisolated from Klebsiella pneumoniae pDT29 contains the open readingframes dhaR, orfY, dhaT, orfX, orfW, dhaB1, dhaB2, and dhaB3, nucleotidethe sequences of which are contained within SEQ ID NO:1. pKP32 containsthe same set of open reading frames as found on pDT29, from the samesource, with the difference that pKP32 lacks the dhaT. pAH48 is thevehicle used for the introduction of DAR1 and GPP2 genes into the hostcell and more specifically comprises the DAR1 and GPP2 genes isolatedfrom Saccharomyces cerevisiae.

Transformation of Suitable Hosts and Expression of Genes for theProduction of 1,3-propanediol

Once suitable cassettes are constructed they are used to transformappropriate host cells. Introduction of the cassette containing thegenes encoding G3PDH, G3P phosphatase, dehydratase, and dehydratasereactivation factor into the host cell may be accomplished by knownprocedures such as by transformation (e.g., using calcium-permeabilizedcells, electroporation), or by transfection using a recombinant phagevirus (Sambrook et al., supra).

In the present invention cassettes were used to transform the E. coli asfully described in the GENERAL METHODS and EXAMPLES.

Mutants

In addition to the cells exemplified, it is contemplated that thepresent method will be able to make use of cells having single ormultiple mutations specifically designed to enhance the production of1,3-propanediol. Cells that normally divert a carbon feed stock intonon-productive pathways, or that exhibit significant cataboliterepression could be mutated to avoid these phenotypic deficiencies. Forexample, many wild type cells are subject to catabolite repression fromglucose and by-products in the media and it is contemplated that mutantstrains of these wild type organisms, capable of 1,3-propanediolproduction that are resistant to glucose repression, would beparticularly useful in the present invention.

Methods of creating mutants are common and well known in the art. Forexample, wild type cells may be exposed to a variety of agents such asradiation or chemical mutagens and then screened for the desiredphenotype. When creating mutations through radiation either ultraviolet(UV) or ionizing radiation may be used. Suitable short wave UVwavelengths for genetic mutations will fall within the range of 200 nmto 300 nm where 254 nm is preferred. UV radiation in this wavelengthprincipally causes changes within nucleic acid sequence from guanidineand cytosine to adenine and thymidine. Since all cells have DNA repairmechanisms that would repair most UV induced mutations, agents such ascaffeine and other inhibitors may be added to interrupt the repairprocess and maximize the number of effective mutations. Long wave UVmutations using light in the 300 nm to 400 nm range are also possiblebut are generally not as effective as the short wave UV light unlessused in conjunction with various activators such as psoralen dyes thatinteract with the DNA.

Mutagenesis with chemical agents is also effective for generatingmutants and commonly used substances include chemicals that affectnonreplicating DNA such as HNO₂ and NH₂OH, as well as agents that affectreplicating DNA such as acridine dyes, notable for causing frameshiftmutations. Specific methods for creating mutants using radiation orchemical agents are well documented in the art. See for example ThomasD. Brock in Biotechnology: A Textbook of Industrial Microbiology, SecondEdition (1989) Sinauer Associates, Inc., Sunderland, Mass., orDeshpande, Mukund V., Appl. Biochem. Biotechnol. 36, 227 (1992), hereinincorporated by reference.

After mutagenesis has occurred, mutants having the desired phenotype maybe selected by a variety of methods. Random screening is most commonwhere the mutagenized cells are selected for the ability to produce thedesired product or intermediate. Alternatively, selective isolation ofmutants can be performed by growing a mutagenized population onselective media where only resistant colonies can develop. Methods ofmutant selection are highly developed and well known in the art ofindustrial microbiology. See for example Brock, Supra; DeMancilha etal., Food Chem. 14, 313 (1984).

The elimination of an undesired enzyme activity may be also accomplishedby disruption of the gene encoding the enzyme. Such methods are known tothose skilled in the art and are exemplified in Example 4 and Example 8.

Alterations in the 1,3-propanediol Production Pathway

Representative Enzyme Pathway. The production of 1,3-propanediol fromglucose can be accomplished by the following series of steps. Thisseries is representative of a number of pathways known to those skilledin the art and is illustrated in FIG. 5. Glucose is converted in aseries of steps by enzymes of the glycolytic pathway to dihydroxyacetonephosphate (DHAP) and 3-phospho-glyceraldehyde (3-PG). Glycerol is thenformed by either hydrolysis of DHAP to dihydroxyacetone (DHA) followedby reduction, or reduction of DHAP to glycerol 3-phosphate (G3P)followed by hydrolysis. The hydrolysis step can be catalyzed by anynumber of cellular phosphatases, which are known to be non-specific withrespect to their substrates, or the activity can be introduced into thehost by recombination. The reduction step can be catalyzed by a NAD⁺ (orNADP⁺) linked host enzyme or the activity can be introduced into thehost by recombination. It is notable that the dha regulon contains aglycerol dehydrogenase (E.C. 1.1.1.6) that catalyzes the reversiblereaction of Equation 3.

Glycerol→3-HPA+H₂O  (Equation 1)

3-HPA+NADH+H⁺→1,3-Propanediol+NAD⁺  (Equation 2)

Glycerol+NAD⁺→DHA+NADH+H⁺  (Equation 3)

Glycerol is converted to 1,3-propanediol via the intermediate3-hydroxy-propionaldehye (3-HPA) as has been described in detail above.The intermediate 3-HPA is produced from glycerol, Equation 1, by adehydratase enzyme that can be encoded by the host or can be introducedinto the host by recombination. This dehydratase can be glyceroldehydratase (E.C. 4.2.1.30), diol dehydratase (E.C. 4.2.1.28) or anyother enzyme able to catalyze this transformation. Glycerol dehydratase,but not diol dehydratase, is encoded by the dha regulon. 1,3-Propanediolis produced from 3-HPA, Equation 2, by a NAD⁺- (or NADP⁺) linked hostenzyme or the activity can be introduced into the host by recombination.This final reaction in the production of 1,3-propanediol can becatalyzed by 1,3-propanediol dehydrogenase (E.C. 1.1.1.202) or otheralcohol dehydrogenases.

Mutations and transformations that affect carbon channeling. A varietyof mutant microorganisms comprising variations in the 1,3-propanediolproduction pathway will be useful in the present invention. For examplethe introduction of a triosephosphate isomerase mutation (tpi-) into themicroorganism of the present invention is an example of the use of amutation to improve the performance by carbon channeling.Triosephosphate isomerase is the enzyme responsible for the conversionof DAHP to 3-phosphoglyceraldehyde, and as such allows the diversion ofcarbon flow from the main pathway form glucose to glycerol and1,3-propanediol (FIG. 5). Thus, the deletion mutation (tpi-) enhancesthe overall metabolic efficiency of the desired pathway over thatdescribed in the art. Similarly, mutations which block alternatepathways for intermediates of the 1,3-propanediol production pathwaywould also be useful to the present invention. For example, theelimination of glycerol kinase prevents glycerol, formed from G3P by theaction of G3P phosphatase, from being re-converted to G3P at the expenseof ATP (FIG. 5). Also, the elimination of glycerol dehydrogenase (forexample, gldA) prevents glycerol, formed from DHAP by the action ofNADH-dependent glycerol-3-phosphate dehydrogenase, from being convertedto dihydroxyacetone (FIG. 5). Mutations can be directed toward astructural gene so as to impair or improve the activity of an enzymaticactivity or can be directed toward a regulatory gene, including promoterregions and ribosome binding sites, so as to modulate the expressionlevel of an enzymatic activity.

It is thus contemplated that transformations and mutations can becombined so as to control particular enzyme activities for theenhancement of 1,3-propanediol production. Thus, it is within the scopeof the present invention to anticipate modifications of a whole cellcatalyst which lead to an increased production of 1,3-propanediol.

The present invention utilizes a preferred pathway for the production of1,3-propanediol from a sugar substrate where the carbon flow moves fromglucose to DHAP, G3P, Glycerol, 3-HPA and finally to 1,3-propanediol.The present production strains have been engineered to maximize themetabolic efficiency of the pathway by incorporating various deletionmutations that prevent the diversion of carbon to non-productivecompounds. Glycerol may be diverted from conversion to 3HPA bytransformation to either DHA or G3P via glycerol dehydrogenase orglycerol kinase as discussed above (FIG. 5). Accordingly, the presentproduction strains contain deletion mutations in the gldA and glpKgenes. Similarly DHAP may be diverted to 3-PG by triosephosphateisomerase, thus the present production microorganism also contains adeletion mutation in this gene. The present method additionallyincorporates a dehydratase enzyme for the conversion of glycerol to3HPA, which functions in concert with the reactivation factor, encodedby orfX and orfZ of the dha regulon (FIG. 5). Although conversion of the3HPA to 1,3-propanediol is typically accomplished via a 1,3-propanedioloxidoreductase, the present method utilizes a non-specific catalyticactivity that produces greater titers and yields of the final product,1,3-propanediol (FIG. 5). In such a process, titers of 1,3-propanediolof at least 10 g/L are achieved, where titers of 200 g/L are expected.

Alternatively, an improved process for 1,3-propanediol production mayutilize glycerol or dihydroxyacetone as a substrate where the pathwaycomprises only the last three substrates, glycerol→3HPA→1,3-propanediol. In such a process, the oxidoreductase is againeliminated in favor of the non-specific catalytic activity, (expected tobe an alcohol dehydrogenase), however the need for deletion mutationsare nullified by the energy considerations of adding glycerol to theculture. In such as process titers of 1,3-propanediol of at least 71 g/Lare achieved where titers of 200 g/L are expected.

Similarly it is within the scope of the invention to provide mutants ofwildtype microorganisms that have been modified by the deletion ormutation of the dhaT activity to create improved 1,3-propandiolproducers. For example, microorganisms, which naturally contain all theelements of the dha regulon, may be manipulated so as to inactivate thedhaT gene encoding the 1,3-propandiol oxidoreductase activity. Thesemicroorganisms will be expected to produce higher yields and titers of1,3-propanediol, mediated by the presence of an endogenous catalyticactivity, expected to be an alcohol dehydrogenase. Examples of suchmicroorganisms include but are not limited to Klebsiella sp.,Citrobacter sp., and Clostridium sp.

Media and Carbon Substrates

Fermentation media in the present invention must contain suitable carbonsubstrates. Suitable substrates may include but are not limited tomonosaccharides such as glucose and fructose, oligosaccharides such aslactose or sucrose, polysaccharides such as starch or cellulose ormixtures thereof and unpurified mixtures from renewable feedstocks suchas cheese whey permeate, cornsteep liquor, sugar beet molasses, andbarley malt. Additionally the carbon substrate may also be one-carbonsubstrates such as carbon dioxide, or methanol for which metabolicconversion into key biochemical intermediates has been demonstrated.Glycerol production from single carbon sources (e.g., methanol,formaldehyde or formate) has been reported in methylotrophic yeasts (K.Yamada et al., Agric. Biol. Chem. 53(2), 541-543 (1989)) and in bacteria(Hunter et. al., Biochemistry 24, 4148-4155 (1985)). Thesemicroorganisms can assimilate single carbon compounds, ranging inoxidation state from methane to formate, and produce glycerol. Thepathway of carbon assimilation can be through ribulose monophosphate,through serine, or through xylulose-momophosphate (Gottschalk, BacterialMetabolism, Second Edition, Springer-Verlag: New York (1986)). Theribulose monophosphate pathway involves the condensation of formate withribulose-5-phosphate to form a 6-carbon sugar that becomes fructose andeventually the three-carbon product glyceraldehyde-3-phosphate.Likewise, the serine pathway assimilates the one-carbon compound intothe glycolytic pathway via methylenetetrahydrofolate.

In addition to one and two carbon substrates, methylotrophicmicroorganisms are also known to utilize a number of othercarbon-containing compounds such as methylamine, glucosamine and avariety of amino acids for metabolic activity. For example,methylotrophic yeast are known to utilize the carbon from methylamine toform trehalose or glycerol (Bellion et al., Microb. Growth C1 Compd.,[Int. Symp.], 7th (1993), 415-32. Editor(s): Murrell, J. Collin; Kelly,Don P. Publisher: Intercept, Andover, UK). Similarly, various species ofCandida will metabolize alanine or oleic acid (Sulter et al., Arch.Microbiol. 153(5), 485-489 (1990)). Hence, it is contemplated that thesource of carbon utilized in the present invention may encompass a widevariety of carbon-containing substrates and will only be limited by thechoice of microorganism or process.

Although it is contemplated that all of the above mentioned carbonsubstrates and mixtures (co-feed) thereof are suitable in the presentinvention, preferred carbon substrates are glucose, fructose, sucrose,or methanol where the process intends to produce an endogenous glycerol,and glycerol or dihydroxyacetone where the process anticipates aglycerol or dihydroxyacetone feed.

In addition to an appropriate carbon source, fermentation media mustcontain suitable minerals, salts, cofactors, buffers and othercomponents, known to those skilled in the art, suitable for the growthof the cultures and promotion of the enzymatic pathway necessary for1,3-propanediol production. Particular attention is given to Co(II)salts and/or vitamin B₁₂ or precursors thereof.

Adenosyl-cobalamin (coenzyme B₁₂) is an essential cofactor fordehydratase activity. Synthesis of coenzyme B₁₂ is found in prokaryotes,some of which are able to synthesize the compound de novo, for example,Escherichia blattae, Klebsiella species, Citrobacter species, andClostridium species, while others can perform partial reactions. E.coli, for example, cannot fabricate the corrin ring structure, but isable to catalyze the conversion of cobinamide to corrinoid and canintroduce the 5′-deoxyadenosyl group. Thus, it is known in the art thata coenzyme B₁₂ precursor, such as vitamin B₁₂, need be provided in E.coli fermentations.

Vitamin B 2 additions to E. coli fermentations may be addedcontinuously, at a constant rate or staged as to coincide with thegeneration of cell mass, or may be added in single or multiple bolusadditions. Preferred ratios of vitamin B₁₂ (mg) fed to cell mass (OD550)are from 0.06 to 0.60. Most preferred ratios of vitamin B₁₂ (mg) fed tocell mass (OD550) are from 0.12 to 0.48.

Although vitamin B₁₂ is added to the transformed E. coli of the presentinvention it is contemplated that other microorganisms, capable of denovo B₁₂ biosynthesis will also be suitable production cells and theaddition of B₁₂ to these microorganisms will be unnecessary.

Culture Conditions:

Typically cells are grown at 35° C. in appropriate media. Preferredgrowth media in the present invention are common commercially preparedmedia such as Luria Bertani (LB) broth, Sabouraud Dextrose (SD) broth orYeast medium (YM) broth. Other defined or synthetic growth media mayalso be used and the appropriate medium for growth of the particularmicroorganism will be known by someone skilled in the art ofmicrobiology or fermentation science. The use of agents known tomodulate catabolite repression directly or indirectly, e.g., cyclicadenosine 2′:3′-monophosphate, may also be incorporated into thereaction media. Similarly, the use of agents known to modulate enzymaticactivities (e.g., methyl viologen) that lead to enhancement of1,3-propanediol production may be used in conjunction with or as analternative to genetic manipulations.

Suitable pH ranges for the fermentation are between pH 5.0 to pH 9.0,where pH 6.0 to pH 8.0 is preferred as the initial condition.

Reactions may be performed under aerobic or anaerobic conditions whereanaerobic or microaerobic conditions are preferred.

Fed-batch fermentations may be performed with carbon feed, for example,glucose, limited or excess.

Batch and Continuous Fermentations:

The present process employs a batch method of fermentation. Classicalbatch fermentation is a closed system where the composition of the mediais set at the beginning of the fermentation and is not subject toartificial alterations during the fermentation. Thus, at the beginningof the fermentation the media is inoculated with the desiredmicroorganism or microorganisms and fermentation is permitted to occuradding nothing to the system. Typically, however, “batch” fermentationis batch with respect to the addition of carbon source and attempts areoften made at controlling factors such as pH and oxygen concentration.In batch systems the metabolite and biomass compositions of the systemchange constantly up to the time the fermentation is stopped. Withinbatch cultures cells moderate through a static lag phase to a highgrowth log phase and finally to a stationary phase where growth rate isdiminished or halted. If untreated, cells in the stationary phase willeventually die. Cells in log phase generally are responsible for thebulk of production of end product or intermediate. A variation on thestandard batch system is the Fed-Batch system.

Fed-Batch fermentation processes are also suitable in the presentinvention and comprise a typical batch system with the exception thatthe substrate is added in increments as the fermentation progresses.Fed-Batch systems are useful when catabolite repression is apt toinhibit the metabolism of the cells and where it is desirable to havelimited amounts of substrate in the media. Measurement of the actualsubstrate concentration in Fed-Batch systems is difficult and istherefore estimated on the basis of the changes of measurable factorssuch as pH, dissolved oxygen and the partial pressure of waste gasessuch as CO₂. Batch and Fed-Batch fermentations are common and well knownin the art and examples may be found in Brock, supra.

Although the present invention is performed in batch mode it iscontemplated that the method would be adaptable to continuousfermentation methods. Continuous fermentation is an open system where adefined fermentation media is added continuously to a bioreactor and anequal amount of conditioned media is removed simultaneously forprocessing. Continuous fermentation generally maintains the cultures ata constant high density where cells are primarily in log phase growth.

Continuous fermentation allows for the modulation of one factor or anynumber of factors that affect cell growth or end product concentration.For example, one method will maintain a limiting nutrient such as thecarbon source or nitrogen level at a fixed rate and allow all otherparameters to moderate. In other systems a number of factors affectinggrowth can be altered continuously while the cell concentration,measured by media turbidity, is kept constant. Continuous systems striveto maintain steady state growth conditions and thus the cell loss due tomedia being drawn off must be balanced against the cell growth rate inthe fermentation. Methods of modulating nutrients and growth factors forcontinuous fermentation processes as well as techniques for maximizingthe rate of product formation are well known in the art of industrialmicrobiology and a variety of methods are detailed by Brock, supra.

It is contemplated that the present invention may be practiced usingbatch, fed-batch or continuous processes and that any known mode offermentation would be suitable. Additionally, it is contemplated thatcells may be immobilized on a substrate as whole cell catalysts andsubjected to fermentation conditions for 1,3-propanediol production.

Identification and purification of 1,3-propanediol:

Methods for the purification of 1,3-propanediol from fermentation mediaare known in the art. For example, propanediols can be obtained fromcell media by subjecting the reaction mixture to extraction with anorganic solvent, distillation, and column chromatography (U.S. Pat. No.5,356,812). A particularly good organic solvent for this process iscyclohexane (U.S. Pat. No. 5,008,473).

1,3-Propanediol may be identified directly by submitting the media tohigh pressure liquid chromatography (HPLC) analysis. Preferred in thepresent invention is a method where fermentation media is analyzed on ananalytical ion exchange column using a mobile phase of 0.01N sulfuricacid in an isocratic fashion.

EXAMPLES General Methods

Procedures for phosphorylations, ligations and transformations are wellknown in the art. Techniques suitable for use in the following examplesmay be found in Sambrook, J. et al., Molecular Cloning: A LaboratoryManual, Second Edition, Cold Spring Harbor Laboratory Press (1989).

Materials and methods suitable for the maintenance and growth ofbacterial cultures are well known in the art. Techniques suitable foruse in the following examples may be found in Manual of Methods forGeneral Bacteriology (Phillipp Gerhardt, R. G. E. Murray, Ralph N.Costilow, Eugene W. Nester, Willis A. Wood, Noel R. Krieg and G. BriggsPhillips, eds), American Society for Microbiology, Washington, D.C.(1994) or Thomas D. Brock in Biotechnology: A Textbook of IndustrialMicrobiology, Second Edition (1989) Sinauer Associates, Inc.,Sunderland, Mass. All reagents and materials used for the growth andmaintenance of bacterial cells were obtained from Aldrich Chemicals(Milwaukee, Wis.), DIFCO Laboratories (Detroit, Mich.), GIBCO/BRL(Gaithersburg, Md.), or Sigma Chemical Company (St. Louis, Mo.) unlessotherwise specified.

The meaning of abbreviations is as follows: “h” means hour(s), “min”means minute(s), “sec” means second(s), “d” means day(s), “mL” meansmilliliters, “L” means liters, 50 amp is 50 μg/mL ampicillin, and LB-50amp is Luria-Bertani broth containing 50 μg/mL ampicillin.

Within the tables the following abbreviations are used. “Con.” isconversion, “Sel.” is selectivity based on carbon, and “nd” is notdetected.

Strains and vectors used and constructed in the following examples arelisted in the chart below:

STRAIN/PLASMID DELETION ORF/GENE KLP23 gldA glpK RJ8m gldA glpK TpipAH48 GPP2 DAR1 pDT29 dhaR orfY dhaT orfX orfW dhaB1 dhaB2 dhaB3 orfZpKP32 dhaR orfY orfX orfW dhaB1 dhaB2 dhaB3 orfZ

Enzyme Assays Assays for Dehydratase Enzymes:

Dehydratase activity in cell-free extracts was determined using eitherglycerol or 1,2-propanediol as substrate. Typically, cell-free extractswere prepared by cell disruption using a french press followed bycentrifugation of the cellular debris. The assay, based on the reactionof aldehydes with methylbenzo-2-thiazolone hydrazone, has been describedby Forage and Foster (Biochim. Biophys. Acta 569, 249 (1979)).

Honda et al. (J. Bacteriol. 143, 1458 (1980)) disclose an assay thatmeasures the reactivation of dehydratases. Dehydratase activity wasdetermined in toluenized whole cells, with and without ATP, using eitherglycerol or 1,2-propanediol as substrate. Reactivation was determined bythe ratio of product formation with versus without the ATP addition.Product formation (3-HPA or propionaldehyde when glycerol or1,2-propanediol is used as substrate, respectively) was measureddirectly, using HPLC, or indirectly, using the methylbenzo-2-thiazolonehydrazone reagent. Alternatively, product formation was determined bycoupling the conversion of the aldehyde to its respective alcohol usinga NADH linked alcohol dehydrogenase and monitoring the disappearance ofNADH.

Assays for 1,3-propanediol Oxidoreductase:

The activity of 1,3-propanediol oxidoreductase, sometimes referred to as1,3-propanediol dehydrogenase, was determined for cell-free extracts insolution or in slab gels using 1,3-propanediol and NAD⁺ as substrateshas been described (Johnson and Lin, J. Bacteriol. 169, 2050 (1987)).Alternatively, the conversion of 3-HPA and NADH to 1,3-propanediol andNAD⁺ was determined by the disappearance of NADH. The slab gel assay hasthe potential advantage of separating the activity of 1,3-propanedioloxidoreductase (dhaT) from that of non-specific alcohol dehydrogenasesby virtue of size separation. The native molecular weights of1,3-propanediol oxidoreductases (dhaT) from Citrobacter freundii,Klebsiella pneumoniae, and Clostridium pasteurianum are unusually large,on the order of 330,000 to 440,000 daltons. Lactobacillus brevis andLactobacillus buchneri contain dehydratase associated 1,3-propanedioloxidoreductases with properties similar to those of known1,3-propanediol oxidoreductases (dhaT).

Assays for Glycerol 3-phosphate Dehydrogenase Activity:

A procedure was used as modified below from a method published by Bellet al. (J. Biol. Chem. 250, 7153 (1975)). This method involvedincubating a cell-free extract sample in a cuvette that contained 0.2 mMNADH, 2.0 mM dihydroxyacetone phosphate (DHAP), and enzyme in 0.1 MTris/HCl, pH 7.5 buffer with 5 mM DTT, in a total volume of 1.0 mL at30° C. A background rate of the reaction of enzyme and NADH was firstdetermined at 340 nm for at least 3 min. The second substrate, DHAP, wassubsequently added and the absorbance change over time was furthermonitored for at least 3 min. G3PDH activity was defined by subtractingthe background rate from the gross rate.

Assay for Glycerol-3-phosphatase Activity:

The assay for enzyme activity was performed by incubating the extractwith an organic phosphate substrate in a bis-Tris or MES and magnesiumbuffer, pH 6.5. The substrate used was either 1-α-glycerol phosphate, ord,1-α-glycerol phosphate. The final concentrations of the reagents inthe assay are: buffer (20 mM, bis-Tris or 50 mM MES); MgCl₂ (10 mM); andsubstrate (20 mM). If the total protein in the sample was low and novisible precipitation occurs with an acid quench, the sample wasconveniently assayed in the cuvette. This method involved incubating anenzyme sample in a cuvette that contained 20 mM substrate (50 μL, 200mM), 50 mM MES, 10 mM MgCl₂, pH 6.5 buffer. The final phosphatase assayvolume was 0.5 mL. The enzyme-containing sample was added to thereaction mixture; the contents of the cuvette were mixed and then thecuvette was placed in a circulating water bath at T=37° C. for 5 to 120min, the length of time depending on whether the phosphatase activity inthe enzyme sample ranged from 2 to 0.02 U/mL. The enzymatic reaction wasquenched by the addition of the acid molybdate reagent (0.4 mL). Afterthe Fiske SubbaRow reagent (0.1 mL) and distilled water (1.5 mL) wereadded, the solution was mixed and allowed to develop. After 10 min, toallow full color development, the absorbance of the samples was read at660 nm using a Cary 219 UV/vis spectrophotometer. The amount ofinorganic phosphate released was compared to a standard curve that wasprepared by using a stock inorganic phosphate solution (0.65 mM) andpreparing 6 standards with final inorganic phosphate concentrationsranging from 0.026 to 0.130 μmol/mL.

Assay for Glycerol Kinase Activity:

An appropriate amount of enzyme, typically a cell-free crude extract,was added to a reaction mixture containing 40 mM ATP, 20 mM MgSO₄, 21 mMuniformly ¹³C labelled glycerol (99%, Cambridge Isotope Laboratories),and 0.1 M Tris-HCl, pH 9 for 75 min at 25° C. The conversion of glycerolto glycerol 3-phosphate was detected by ¹³C-NMR (125 MHz): glycerol(63.11 ppm, δ, J=41 Hz and 72.66 ppm, t, J=41 Hz); glycerol 3-phosphate(62.93 ppm, δ, J=41 Hz; 65.31 ppm, br d, J=43 Hz; and 72.66 ppm, dt,J=6.41 Hz).

NADH-Linked Glycerol Dehydrogenase Assay:

NADH-linked glycerol dehydrogenase activity (gldA) in cell-free extractsfrom E. coli strains was determined after protein separation bynon-denaturing polyacrylamide gel electrophoresis. The conversion ofglycerol plus NAD⁺ to dihydroxyacetone plus NADH was coupled with theconversion of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazoliumbromide (MTT) to a deeply colored formazan, using phenazine methosulfate(PMS) as mediator (Tang et al., J. Bacteriol. 140, 182 (1997)).

Electrophoresis was performed in duplicate by standard procedures usingnative gels (8-16% TG, 1.5 mm, 15 lane gels from Novex, San Diego,Calif.). Residual glycerol was removed from the gels by washing 3× with50 mM Tris or potassium carbonate buffer, pH 9 for 10 min. The duplicategels were developed, with and without glycerol (approximately 0.16 Mfinal concentration), in 15 mL of assay solution containing 50 mM Trisor potassium carbonate, pH 9, 60 mg ammonium sulfate, 75 mg NAD⁺, 1.5 mgMTT, and 0.5 mg PMS.

The presence or absence of NADH-linked glycerol dehydrogenase activityin E. coli strains (gldA) was also determined, following polyacrylamidegel electrophoresis, by reaction with polyclonal antibodies raised topurified K. pneumoniae glycerol dehydrogenase (dhaD).

Isolation and identification of 1,3-propanediol:

The conversion of glycerol to 1,3-propanediol was monitored by HPLC.Analyses were performed using standard techniques and materialsavailable to one of skill in the art of chromatography. One suitablemethod utilized a Waters Maxima 820 HPLC system using UV (210 nm) and RIdetection. Samples were injected onto a Shodex SH-1011 column (8 mm×300mm, purchased from Waters, Milford, Mass.) equipped with a ShodexSH-1011P precolumn (6 mm×50 mm), temperature controlled at 50° C., using0.01 NH₂SO₄ as mobile phase at a flow rate of 0.5 mL/min. Whenquantitative analysis was desired, samples were prepared with a knownamount of trimethylacetic acid as external standard. Typically, theretention times of glucose (RI detection), glycerol, 1,3-propanediol (RIdetection), and trimethylacetic acid (UV and RI detection) were 15.27min, 20.67 min, 26.08 min, and 35.03 min, respectively.

Production of 1,3-propanediol was confirmed by GC/MS. Analyses wereperformed using standard techniques and materials available to one ofskill in the art of GC/MS. One suitable method utilized a HewlettPackard 5890 Series II gas chromatograph coupled to a Hewlett Packard5971 Series mass selective detector (EI) and a HP-INNOWax column (30 mlength, 0.25 mm i.d., 0.25 micron film thickness). The retention timeand mass spectrum of 1,3-propanediol generated were compared to that ofauthentic 1,3-propanediol (m/e: 57, 58).

An alternative method for GC/MS involved derivatization of the sample.To 1.0 mL of sample (e.g., culture supernatant) was added 30 μL ofconcentrated (70% v/v) perchloric acid. After mixing, the sample wasfrozen and lyophilized. A 1:1 mixture ofbis(trimethylsilyl)trifluoroacetamide:pyridine (300 μL) was added to thelyophilized material, mixed vigorously and placed at 65° C. for one h.The sample was clarified of insoluble material by centrifugation. Theresulting liquid partitioned into two phases, the upper of which wasused for analysis. The sample was chromatographed on a DB-5 column (48m, 0.25 mm I.D., 0.25 μm film thickness; from J&W Scientific) and theretention time and mass spectrum of the 1,3-propanediol derivativeobtained from culture supernatants were compared to that obtained fromauthentic standards. The mass spectra of TMS-derivatized 1,3-propanediolcontains the characteristic ions of 205, 177, 130 and 115 AMU.

Cell Lysis:

Cell lysis was estimated by measuring the extracellular soluble proteinconcentration in the fermentation broth. Fermenter samples werecentrifuged in a desktop centrifuge (typically, 3-5 min at 12,000 rpm inan Eppendorf, Model 5415C micro centrifuge) in order to separate cells.The resulting supernatant was analyzed for protein concentration by theBradford method using a commercially available reagent (Bio-Rad ProteinAssay, Bio-Rad, Hercules, Calif.).

Viability:

Cell viability was determined by plating, at appropriate dilutions,cells obtained from the fermenter on non-selective LB agar plates. Cellviability between fermenter experiments is compared by using the ratioof viable cells per mL of fermenter broth divided by OD550 (AU).

Example 1 Cloning and Transformation of E. Coli Host Cells with CosmidDNA for the Expression of 1,3-Propanediol Media:

Synthetic S12 medium was used in the screening of bacterialtransformants for the ability to make 1,3-propanediol. S12 mediumcontains: 10 mM ammonium sulfate, 50 mM potassium phosphate buffer, pH7.0, 2 mM MgCl₂, 0.7 mM CaCl₂, 50 μM MnCl₂, 1 μM FeCl₃, 1 M ZnCl, 1.7 μMCuSO₄, 2.5 μM CoCl₂, 2.4 μM Na₂MoO₄, and 2 μM thiamine hydrochloride.

Medium A used for growth and fermentation consisted of: 10 mM ammoniumsulfate; 50 mM MOPS/KOH buffer, pH 7.5; 5 mM potassium phosphate buffer,pH 7.5; 2 mM MgCl₂; 0.7 mM CaCl₂; 50 μM MnCl₂; 1 μM FeCl₃; 1 μM ZnCl;1.72 μM CuSO₄; 2.53 μM CoCl₂; 2.42 μM Na₂MoO₄; 2 μM thiaminehydrochloride; 0.01% yeast extract; 0.01% casamino acids; 0.8 μg/mLvitamin B₁₂; and 50 μg/mL amp. Medium A was supplemented with either0.2% glycerol or 0.2% glycerol plus 0.2% D-glucose as required.

Cells:

Klebsiella pneumoniae. ECL2106 (Ruch et al., J. Bacteriol. 124, 348(1975)), also known in the literature as K. aerogenes or Aerobacteraerogenes, was obtained from E. C. C. Lin (Harvard Medical School,Cambridge, Mass.) and was maintained as a laboratory culture.

Klebsiella pneumoniae. ATCC 25955 was purchased from American TypeCulture Collection (Manassas, Va.).

E. coli DH5α was purchased from Gibco/BRL and was transformed with thecosmid DNA isolated from Klebsiella pneumoniae. ATCC 25955 containing agene coding for either a glycerol or diol dehydratase enzyme. Cosmidscontaining the glycerol dehydratase were identified as pKP1 and pKP2 andcosmid containing the diol dehydratase enzyme were identified as pKP4.Transformed DH5α cells were identified as DH5α-pKP1, DH5α-pKP2, andDH5α-pKP4.

E. coli ECL707 (Sprenger et al., J. Gen. Microbiol. 135, 1255 (1989))was obtained from E. C. C. Lin (Harvard Medical School, Cambridge,Mass.) and was similarly transformed with cosmid DNA from Klebsiellapneumoniae. These transformants were identified as ECL707-pKP1 andECL707-pKP2, containing the glycerol dehydratase gene and ECL707-pKP4containing the diol dehydratase gene.

E. coli AA200 containing a mutation in the tpi gene (Anderson et al., J.Gen. Microbiol. 62, 329 (1970)) was purchased from the E. coli GeneticStock Center, Yale University (New Haven, Conn.) and was transformedwith Klebsiella cosmid DNA to give the recombinant microorganismsAA200-pKP1 and AA200-pKP2, containing the glycerol dehydratase gene, andAA200-pKP4, containing the diol dehydratase gene.

DH5α:

Six transformation plates containing approximately 1,000 colonies of E.coli XL1-Blue MR transfected with K. pneumoniae. DNA were washed with 5mL LB medium and centrifuged. The bacteria were pelleted and resuspendedin 5 mL LB medium+glycerol. An aliquot (50 μL) was inoculated into a 15mL tube containing S12 synthetic medium with 0.2% glycerol+400 ng per mLof vitamin B₁₂+0.001% yeast extract+50 amp. The tube was filled with themedium to the top and wrapped with parafilm and incubated at 30° C. Aslight turbidity was observed after 48 h. Aliquots, analyzed for productdistribution as described above at 78 h and 132 h, were positive for1,3-propanediol, the later time points containing increased amounts of1,3-propanediol.

The bacteria, testing positive for 1,3-propanediol production, wereserially diluted and plated onto LB-50 amp plates in order to isolatesingle colonies. Forty-eight single colonies were isolated and checkedagain for the production of 1,3-propanediol. Cosmid DNA was isolatedfrom 6 independent clones and transformed into E. coli strain DH5α. Thetransformants were again checked for the production of 1,3-propanediol.Two transformants were characterized further and designated as DH5α-pKP1and DH5α-pKP2.

A 12.1 kb EcoRI-SalI fragment from pKP1, subcloned into pIBI31 (IBIBiosystem, New Haven, Conn.), was sequenced and termed pHK28-26 (SEQ IDNO:1). Sequencing revealed the loci of the relevant open reading framesof the dha operon encoding glycerol dehydratase and genes necessary forregulation. Referring to SEQ ID NO:1, a fragment of the open readingframe for dhaK1 encoding dihydroxyacetone kinase is found at bases 1-399(complement); the open reading frame dhaD encoding glyceroldehydrogenase is found at bases 1010-2107; the open reading frame dhaRencoding the repressor is found at bases 2209-4134; the open readingframe orfW, encoding a protein of unknown function is found at bases4112-4642 (complement); the open reading frame orfX encoding adehydratase reactivation protein is found at bases 4643-4996(complement); the open reading frame dhaT encoding 1,3-propanedioloxidoreductase is found at bases 5017-6180 (complement); the openreading frame orfY, encoding a protein of unknown function is found atbases 6202-6630 (complement); the open reading frame dhaB1 encoding thealpha subunit glycerol dehydratase is found at bases 7044-8711; the openreading frame dhaB2 encoding the beta subunit glycerol dehydratase isfound at bases 8724-9308; the open reading frame dhaB3 encoding thegamma subunit glycerol dehydratase is found at bases 9311-9736; the openreading frame dhaBX, encoding a dehydratase reactivation protein isfound at bases 9749-11572; and a fragment of the open reading frame forglpF encoding a glycerol uptake facilitator protein is found at bases11626-12145.

Single colonies of E. coli XL1-Blue MR transfected with packaged cosmidDNA from K. pneumoniae were inoculated into microtiter wells containing200 μL of S15 medium (ammonium sulfate, 10 mM; potassium phosphatebuffer, pH 7.0, 1 mM; MOPS/KOH buffer, pH 7.0, 50 mM; MgCl₂, 2 mM;CaCl₂, 0.7 mM; MnCl₂, 50 μM; FeCl₃, 1 μM; ZnCl, 1 μM; CuSO₄, 1.72 μM;CoCl₂, 2.53 μM; Na₂MoO₄, 2.42 μM; and thiamine hydrochloride, 2 μM)+0.2%glycerol+400 ng/mL of vitamin B₁₂+0.001% yeast extract+50 μg/mLampicillin. In addition to the microtiter wells, a master platecontaining LB-50 amp was also inoculated. After 96 h, 100 μL waswithdrawn and centrifuged in a Rainin microfuge tube containing a 0.2micron nylon membrane filter. Bacteria were retained and the filtratewas processed for HPLC analysis. Positive clones demonstrating1,3-propanediol production were identified after screening approximately240 colonies. Three positive clones were identified, two of which hadgrown on LB-50 amp and one of which had not. A single colony, isolatedfrom one of the two positive clones grown on LB-50 amp and verified forthe production of 1,3-propanediol, was designated as pKP4. Cosmid DNAwas isolated from E. coli strains containing pKP4 and E. coli strainDH5α was transformed. An independent transformant, designated asDH5α-pKP4, was verified for the production of 1,3-propanediol.

ECL707:

E. coli strain ECL707 was transformed with cosmid K. pneumoniae. DNAcorresponding to one of pKP1, pKP2, pKP4 or the Supercos vector aloneand named ECL707-pKP1, ECL707-pKP2, ECL707-pKP4, and ECL707-sc,respectively. ECL707 is defective in glpK, gld, and ptsD which encodethe ATP-dependent glycerol kinase, NAD⁺-linked glycerol dehydrogenase,and enzyme II for dihydroxyacetone of the phosphoenolpyruvate-dependentphosphotransferase system, respectively.

Twenty single colonies of each cosmid transformation and five of theSupercos vector alone (negative control) transformation, isolated fromLB-50 amp plates, were transferred to a master LB-50 amp plate. Theseisolates were also tested for their ability to convert glycerol to1,3-propanediol in order to determine if they contained dehydrataseactivity. The transformants were transferred with a sterile toothpick tomicrotiter plates containing 200 μL of Medium A supplemented with either0.2% glycerol or 0.2% glycerol plus 0.2% D-glucose. After incubation for48 h at 30° C., the contents of the microtiter plate wells were filteredthrough a 0.45 micron nylon filter and chromatographed by HPLC. Theresults of these tests are given in Table 2.

TABLE 2 Conversion of glycerol to 1,3-propanediol by transformed ECL707transformant glycerol* glycerol plus glucose* ECL707-pKP1 19/20 19/20ECL707-pKP2 18/20 20/20 ECL707-pKP4  0/20 20/20 ECL707-sc 0/5 0/5*(Number of positive isolates/number of isolates tested)

AA200:

E. coli strain AA200 was transformed with cosmid K. pneumoniae. DNAcorresponding to one of pKP1, pKP2, pKP4 and the Supercos vector aloneand named AA200-pKP1, AA200-pKP2, AA200-pKP4, and AA200-sc,respectively. Strain AA200 is defective in triosephosphate isomerase(tpi-).

Twenty single colonies of each cosmid transformation and five of theempty vector transformation were isolated and tested for their abilityto convert glycerol to 1,3-propanediol as described for E. coli strainECL707. The results of these tests are given in Table 3.

TABLE 3 Conversion of glycerol to 1,3-propanediol by transformed AA200transformant glycerol* glycerol plus glucose* AA200-pKP1 17/20 17/20AA200-pKP2 17/20 17/20 AA200-pKP4  2/20 16/20 AA200-sc 0/5 0/5 *(Numberof positive isolates/number of isolates tested)

Example 2 Engineering of Glycerol Kinase Mutants of E. Coli FM5 forProduction of Glycerol from Glucose

Construction of Integration Plasmid for Glycerol Kinase Gene Replacementin E. coli FM5:

E. coli FM5 (ATCC 53911) genomic DNA was prepared using the Puregene DNAIsolation Kit (Gentra Systems, Minneapolis, Minn.). A 1.0 kb DNAfragment containing partial glpF and glycerol kinase (glpK) genes wasamplified by PCR (Mullis and Faloona, Methods Enzymol. 155, 335 (1987))from FM5 genomic DNA using primers SEQ ID NO:2 and SEQ ID NO:3. A 1.1 kbDNA fragment containing partial glpK and glpX genes was amplified by PCRfrom FM5 genomic DNA using primers SEQ ID NO:4 and SEQ ID NO:5. A MunIsite was incorporated into primer SEQ ID NO:4. The 5′ end of primer SEQID NO:4 was the reverse complement of primer SEQ ID NO:3 to enablesubsequent overlap extension PCR. The gene splicing by overlap extensiontechnique (Horton et al., BioTechniques 8, 528 (1990)) was used togenerate a 2.1 kb fragment by PCR using the above two PCR fragments astemplates and primers SEQ ID NO:2 and SEQ ID NO:5. This fragmentrepresented a deletion of 0.8 kb from the central region of the 1.5 kbglpK gene. Overall, this fragment had 1.0 kb and 1.1 kb flanking regionson either side of the MunI cloning site (within the partial glpK) toallow for chromosomal gene replacement by homologous recombination.

The above 2.1 kb PCR fragment was blunt-ended (using mung bean nuclease)and cloned into the pCR-Blunt vector using the Zero Blunt PCR CloningKit (Invitrogen, San Diego, Calif.) to yield the 5.6 kb plasmid pRN100containing kanamycin and Zeocin resistance genes. The 1.2 kb HincIIfragment from pLoxCat1 (unpublished results), containing achloramphenicol-resistance gene flanked by bacteriophage P1 loxP sites(Snaith et al., Gene 166, 173 (1995)), was used to interrupt the glpKfragment in plasmid pRN100 by ligating it to MunI-digested (andblunt-ended) plasmid pRN100 to yield the 6.9 kb plasmid pRN101-1. A 376bp fragment containing the R6K origin was amplified by PCR from thevector pGP704 (Miller and Mekalanos, J. Bacteriol. 170, 2575-2583(1988)) using primers SEQ ID NO:6 and SEQ ID NO:7, blunt-ended, andligated to the 5.3 kb Asp718-AatII fragment (which was blunt-ended) frompRN101-1 to yield the 5.7 kb plasmid pRN102-1 containing kanamycin andchloramphenicol resistance genes. Substitution of the ColE1 originregion in pRN101-1 with the R6K origin to generate pRN102-1 alsoinvolved deletion of most of the Zeocin resistance gene. The host forpRN102-1 replication was E. coli SY327 (Miller and Mekalanos, J.Bacteriol. 170, 2575-2583 (1988)) which contains the pir gene necessaryfor the function of the R6K origin.

Engineering of Glycerol Kinase Mutant RJF10m with ChloramphenicolResistance Gene Interrupt:

E. coli FM5 was electrotransformed with the non-replicative integrationplasmid pRN102-1 and transformants that were chloramphenicol-resistant(12.5 μg/mL) and kanamycin-sensitive (30 μg/mL) were further screenedfor glycerol non-utilization on M9 minimal medium containing 1 mMglycerol. An EcoRI digest of genomic DNA from one such mutant, RJF10m,when probed with the intact glpK gene via Southern analysis (Southern,J. Mol. Biol. 98, 503-517 (1975)) indicated that it was adouble-crossover integrant (glpK gene replacement) since the twoexpected 7.9 kb and 2.0 kb bands were observed, owing to the presence ofan additional EcoRI site within the chloramphenicol resistance gene. Thewild-type control yielded the single expected 9.4 kb band. A ¹³C NMRanalysis of mutant RJF10m confirmed that it was incapable of converting¹³C-labeled glycerol and ATP to glycerol-3-phosphate. This glpK mutantwas further analyzed by genomic PCR using primer combinations SEQ IDNO:8 and SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11, and SEQ ID NO:8 andSEQ ID NO:11 which yielded the expected 2.3 kb, 2.4 kb, and 4.0 kb PCRfragments respectively. The wild-type control yielded the expected 3.5kb band with primers SEQ ID NO:8 and SEQ ID NO:11. The glpK mutantRJF10m was electrotransformed with plasmid pAH48 to allow glycerolproduction from glucose. The glpK mutant E. coli RJF10m has beendeposited with ATCC under the terms of the Budapest Treaty on 24 Nov.1997.

Engineering of Glycerol Kinase Mutant RJF10 with ChloramphenicolResistance Gene Interrupt Removed:

After overnight growth on YENB medium (0.75% yeast extract, 0.8%nutrient broth) at 37° C., E. coli RJF10m in a water suspension waselectrotransformed with plasmid pJW168 (unpublished results), whichcontained the bacteriophage P1 Cre recombinase gene under the control ofthe IPTG-inducible lacUV5 promoter, a temperature-sensitive pSC101replicon, and an ampicillin resistance gene. Upon outgrowth in SOCmedium at 30° C., transformants were selected at 30° C. (permissivetemperature for pJW168 replication) on LB agar medium supplemented withcarbenicillin (50 μg/mL) and IPTG (1 mM). Two serial overnight transfersof pooled colonies were carried out at 30° C. on fresh LB agar mediumsupplemented with carbenicillin and IPTG in order to allow excision ofthe chromosomal chloramphenicol resistance gene via recombination at theloxP sites mediated by the Cre recombinase (Hoess and Abremski, J. Mol.Biol. 181, 351-362 (1985)). Resultant colonies were replica-plated on toLB agar medium supplemented with carbenicillin and IPTG and LB agarsupplemented with chloramphenicol (12.5 μg/mL) to identify colonies thatwere carbenicillin-resistant and chloramphenicol-sensitive indicatingmarker gene removal. An overnight 30° C. culture of one such colony wasused to inoculate 10 mL of LB medium. Upon growth at 30° C. to OD (600nm) of 0.6 AU, the culture was incubated at 37° C. overnight. Severaldilutions were plated on prewarmed LB agar medium and the platesincubated overnight at 42° C. (the non-permissive temperature for pJW168replication). Resultant colonies were replica-plated on to LB agarmedium and LB agar medium supplemented with carbenicillin (75 μg/mL) toidentify colonies that were carbenicillin-sensitive indicating loss ofplasmid pJW168. One such glpK mutant, RJF10, was further analyzed bygenomic PCR using primers SEQ ID NO:8 and SEQ ID NO:11 and yielded theexpected 3.0 kb band confirming marker gene excision. Glycerolnon-utilization by mutant RJF10 was confirmed by lack of growth on M9minimal medium containing 1 mM glycerol. The glpK mutant RJF10 waselectrotransformed with plasmid pAH48 to allow glycerol production fromglucose.

Example 3 Construction of E. Coli Strain with gldA Gene Knockout

The gldA gene was isolated from E. coli by PCR (K. B. Mullis and F. A.Faloona, Meth. Enzymol. 155, 335-350 (1987)) using primers SEQ ID NO:12and SEQ ID NO:13, which incorporate terminal Sph1 and Xba1 sites,respectively, and cloned (T. Maniatis (1982) Molecular Cloning: ALaboratory Manual. Cold Spring Harbor, Cold Spring Harbor, N.Y.) betweenthe Sph1 and Xba1 sites in pUC18, to generate pKP8. pKP8 was cut at theunique Sal1 and Nco1 sites within the gldA gene, the ends flushed withKlenow and religated, resulting in a 109 bp deletion in the middle ofgldA and regeneration of a unique Sal1 site, to generate pKP9. A 1.4 kbDNA fragment containing the gene conferring kanamycin resistance (kan),and including about 400 bps of DNA upstream of the translational startcodon and about 100 bps of DNA downstream of the translational stopcodon, was isolated from pET-28a(+) (Novagen, Madison, Wis.) by PCRusing primers SEQ ID NO:14 and SEQ ID NO:15, which incorporate terminalSal1 sites, and subcloned into the unique Sal1 site of pKP9, to generatepKP13. A 2.1 kb DNA fragment beginning 204 bps downstream of the gldAtranslational start codon and ending 178 bps upstream of the gldAtranslational stop codon, and containing the kan insertion, was isolatedfrom pKP13 by PCR using primers SEQ ID NO:16 and SEQ ID NO:17, whichincorporate terminal Sph1 and Xba1 sites, respectively, was subclonedbetween the Sph1 and Xba1 sites in pMAK705 (Genencor International, PaloAlto, Calif.), to generate pMP33. E. coli FM5 was transformed with pMP33and selected on 20 μg/mL kan at 30° C., which is the permissivetemperature for pMAK705 replication. One colony was expanded overnightat 30° C. in liquid media supplemented with 20 μg/mL kan. Approximately32,000 cells were plated on 20 μg/mL kan and incubated for 16 h at 44°C., which is the restrictive temperature for pMAK705 replication.Transformants growing at 44° C. have plasmid integrated into thechromosome, occurring at a frequency of approximately 0.0001. PCR andSouthern blot (E. M. Southern, J. Mol. Biol. 98, 503-517 (1975))analyses were used to determine the nature of the chromosomalintegration events in the transformants. Western blot analysis (Towbinet al., Proc. Natl. Acad. Sci. 76, 4350 (1979)) was used to determinewhether glycerol dehydrogenase protein, the product of gldA, is producedin the transformants. An activity assay was used to determine whetherglycerol dehydrogenase activity remained in the transformants. Activityin glycerol dehydrogenase bands on native gels was determined bycoupling the conversion of glycerol plus NAD⁺ to dihydroxyacetone plusNADH to the conversion of a tetrazolium dye, MTT[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] to adeeply colored formazan, with phenazine methosulfate as mediator.Glycerol dehydrogenase also requires the presence of 30 mM ammoniumsulfate and 100 mM Tris, pH 9 (Tang et al., J. Bacteriol. 140, 182(1997)). Of 8 transformants analyzed, 6 were determined to be gldAknockouts. E. coli MSP33.6 has been deposited with ATCC under the termsof the Budapest Treaty on 24 Nov. 1997.

Example 4 Construction of an E. Coli Strain with glpK and gldA GeneKnockouts

A 1.6 kb DNA fragment containing the gldA gene and including 228 bps ofDNA upstream of the translational start codon and 220 bps of DNAdownstream of the translational stop codon was isolated from E. coli byPCR using primers SEQ ID NO:18 and SEQ ID NO:19, which incorporateterminal Sph1 and Xba1 sites, respectively, and cloned between the Sph1and Xba1 sites of pUC18, to generate pQN2. pQN2 was cut at the uniqueSal1 and Nco1 sites within the gldA gene, the ends flushed with Klenowand religated, resulting in a 109 bps deletion in the middle of gldA andregeneration of a unique Sal1 site, to generate pQN4. A 1.2 kb DNAfragment containing the gene conferring kanamycin resistance (kan), andflanked by loxP sites was isolated from pLoxKan2 (GenencorInternational, Palo Alto, Calif.) as a Stu1/Xho1 fragment, the endsflushed with Klenow, and subcloned into pQN4 at the Sal1 site afterflushing with Klenow, to generate pQN8. A 0.4 kb DNA fragment containingthe R6K origin of replication was isolated from pGP704 (Miller andMekalanos, J. Bacteriol. 170, 2575-2583 (1988)) by PCR using primers SEQID NO:20 and SEQ ID NO:21, which incorporate terminal Sph1 and Xba1sites, respectively, and ligated to the 2.8 kb Sph1/Xba1 DNA fragmentcontaining the gldA::kan cassette from pQN8, to generate pKP22. A 1.0 kbDNA fragment containing the gene conferring chloramphenicol resistance(cam), and flanked by loxP sites was isolated from pLoxCat2 (GenencorInternational, Palo Alto, Calif.) as an Xba1 fragment, and subclonedinto pKP22 at the Xba1 site, to generate pKP23. E. coli strain RJF10(see Example 2), which is glpK-, was transformed with pKP23 andtransformants with the phenotype kanRcamS were isolated, indicatingdouble crossover integration, which was confirmed by southern blotanalysis. Glycerol dehydrogenase gel activity assays (as described inExample 3) demonstrated that active glycerol dehydrogenase was notpresent in these transformants. The kan marker was removed from thechromosome using the Cre-producing plasmid pJW168, as described inExample 2, to produce strain KLP23. Several isolates with the phenotypekanS demonstrated no glycerol dehydrogenase activity, and southern blotanalysis confirmed loss of the kan marker.

Example 5 Plasmid Construction and Strain Construction for theExpression of Glycerol 3-Phosphate Dehydrogenase (DAR1) and/or Glycerol3-Phosphatase (GPP2)

Construction of Expression Cassettes for Glycerol 3-phosphatase (GPP2):

The Saccharomyces cerevisiae chromosomeV lamda clone 6592 (GenBank,accession # U18813×11) was obtained from ATCC. The glycerol 3-phosphatephosphatase gene (GPP2) was cloned by cloning from the lamda clone astarget DNA using synthetic primers (SEQ ID NO:22 with SEQ ID NO:23)incorporating an BamHI-RBS-XbaI site at the 5′ end and a SmaI site atthe 3′ end. The product was subcloned into pCR-Script (Stratagene,Madison, Wis.) at the SrfI site to generate the plasmid pAH15 containingGPP2. The plasmid pAH15 contains the GPP2 gene in the inactiveorientation for expression from the lac promoter in pCR-Script SK+. TheBamHI-SmaI fragment from pAH15 containing the GPP2 gene was insertedinto pBlueScriptII SK+ to generate plasmid pAH19. The pAH19 contains theGPP2 gene in the correct orientation for expression from the lacpromoter. The XbaI-PstI fragment from pAH19 containing the GPP2 gene wasinserted into pPHOX2 to create plasmid pAH21. The pAH21/DH5α is theexpression plasmid.

Construction of Expression Cassettes for Glycerol 3-phosphateDehydrogenase (DAR1):

DAR1 was isolated by PCR cloning from genomic S. cerevisiae DNA usingsynthetic primers (SEQ ID NO:24 with SEQ ID NO:25). Successful PCRcloning places an NcoI site at the 5′ end of DAR1 where the ATG withinNcoI is the DAR1 initiator methionine. At the 3′ end of DAR1 a BamHIsite is introduced following the translation terminator. The PCRfragments were digested with NcoI+BamHI and cloned into the same siteswithin the expression plasmid pTrc99A (Pharmacia, Piscataway, N.J.) togive pDAR1A.

In order to create a better ribosome binding site at the 5′ end of DAR1,an SpeI-RBS-NcoI linker obtained by annealing synthetic primers (SEQ IDNO:26 with SEQ ID NO:27) was inserted into the NcoI site of pDAR1A tocreate pAH40. Plasmid pAH40 contains the new RBS and DAR1 gene in thecorrect orientation for expression from the trc promoter of pTrc99A(Pharmacia, Piscataway, N.J.). The NcoI-BamHI fragment from pDAR1A andan second set of SpeI-RBS-NcoI linker obtained by annealing syntheticprimers (SEQ ID NO:28 with SEQ ID NO:29) was inserted into theSpeI-BamHI site of pBC-SK+ (Stratagene, Madison, Wis.) to create plasmidpAH42. The plasmid pAH42 contains a chloramphenicol resistant gene.

Construction of Expression Cassettes for DAR1 and GPP2:

Expression cassettes for DAR1 and GPP2 were assembled from theindividual DAR1 and GPP2 subclones described above using standardmolecular biology methods. The BamHI-PstI fragment from pAH19 containingthe ribosomal binding site (RBS) and GPP2 gene was inserted into pAH40to create pAH43. The BamHI-PstI fragment from pAH19 containing the RBSand GPP2 gene was inserted into pAH42 to create pAH45.

The ribosome binding site at the 5′ end of GPP2 was modified as follows.A BamHI-RBS-SpeI linker, obtained by annealing synthetic primersGATCCAGGAAACAGA (SEQ ID NO:30) with CTAGTCTGTTTCCTG (SEQ ID NO:31) tothe XbaI-PstI fragment from pAH19 containing the GPP2 gene, was insertedinto the BamHI-PstI site of pAH40 to create pAH48. Plasmid pAH48contains the DAR1 gene, the modified RBS, and the GPP2 gene in thecorrect orientation for expression from the trc promoter of pTrc99A(Pharmacia, Piscataway, N.J.).

Transformation of E. coli:

The plasmids described here were transformed into E. coli DH5α, FM5 andKLP23 using standard molecular biology techniques. The transformantswere verified by their DNA RFLP pattern.

Example 6 Construction of Expression Plasmids for Use in Transformationof Escherichia Coli with Genes from the Klebsiella Pneumoniae dhaRegulon

Construction of the Expression Vector pTacIQ:

The E. coli expression vector pTacIQ was prepared by inserting lacIqgene (Farabaugh, Nature 274(5673), 765-769 (1978)) and tac promoter(Amann et al., Gene 25, 167-178 (1983)) into the restrictionendonuclease site EcoRI of pBR322 (Sutcliffe, Cold Spring Harb. Symp.Quant. Biol. 43, 77-90 (1979)). A multiple cloning site and terminatorsequence (SEQ ID NO:32) replaces the pBR322 sequence from EcoRI to SphI.

Subcloning the Glycerol Dehydratase Genes (dhaB1,2,3, X):

The open reading frame for the dhaB3 gene was amplified from pHK28-26 byPCR using primers (SEQ ID NO:33 and SEQ ID NO:34) incorporating an EcoRIsite at the 5′ end and a Xba1 site at the 3′ end. The product wassubcloned into pLitmus29 (New England Biolab, Inc., Beverly, Mass.) togenerate the plasmid pDHAB3 containing dhaB3.

The region containing the entire coding region for dhaB1, dhaB2, dhaB3and dhaBX of the dhaB operon from pHK28-26 was cloned intopBluescriptIIKS+ (Stratagene, La Jolla, Calif.) using the restrictionenzymes KpnI and EcoRI to create the plasmid pM7.

The dhaBX gene was removed by digesting plasmid pM7 with ApaI and XbaI,purifying the 5.9 kb fragment and ligating it with the 325-bp ApaI-XbaIfragment from plasmid pDHAB3 to create pM11 containing dhaB1, dhaB2 anddhaB3.

The open reading frame for the dhaB1 gene was amplified from pHK28-26 byPCR using primers (SEQ ID NO:35 and SEQ ID NO:36) incorporating aHindIII site and a consensus ribosome binding site at the 5′ end and aXbaI site at the 3′ end. The product was subcloned into pLitmus28 (NewEngland Biolab, Inc., Beverly, Mass.) to generate the plasmid pDT1containing dhaB1.

A NotI-XbaI fragment from pM11 containing part of the dhaB1 gene, thedhaB2 gene and the dhaB3 gene was inserted into pDT1 to create the dhaBexpression plasmid, pDT2. The HindIII-XbaI fragment containing thedhaB(1,2,3) genes from pDT2 was inserted into pTacIQ to create pDT3.

Subcloning the 1,3-propanediol Dehydrogenase Gene (dhaT):

The KpnI-SacI fragment of pHK28-26, containing the 1,3-propanedioldehydrogenase (dhaT) gene, was subcloned into pBluescriptII KS+ creatingplasmid pAH1. The dhaT gene was amplified by PCR from pAH1 as templateDNA and synthetic primers (SEQ ID NO:37 with SEQ ID NO:38) incorporatingan Xba1 site at the 5′ end and a BamHI site at the 3′ end. The productwas subcloned into pCR-Script (Stratagene) at the SrfI site to generatethe plasmids pAH4 and pAH5 containing dhaT. The plasmid pAH4 containsthe dhaT gene in the right orientation for expression from the lacpromoter in pCR-Script and pAH5 contains dhaT gene in the oppositeorientation. The XbaI-BamHI fragment from pAH4 containing the dhaT genewas inserted into pTacIQ to generate plasmid pAH8. The HindIII-BamHIfragment from pAH8 containing the RBS and dhaT gene was inserted intopBluescriptIIKS+ to create pAH11.

Construction of an Expression Cassette for dhaT and dhaB(1,2,3):

An expression cassette for dhaT and dhaB(1,2,3) was assembled from theindividual dhaB(1,2,3) and dhaT subclones described previously usingstandard molecular biology methods. A SpeI-SacI fragment containing thedhaB(1,2,3) genes from pDT3 was inserted into pAH11 at the SpeI-SacIsites to create pAH24. A SalI-XbaI linker (SEQ ID NO:39 and SEQ IDNO:40) was inserted into pAH5 that was digested with the restrictionenzymes SalI-XbaI to create pDT16. The linker destroys the Xba1 site.The 1 kb SalI-MluI fragment from pDT16 was then inserted into pAH24replacing the existing SalI-MluI fragment to create pDT18. pDT21 wasconstructed by inserting the SalI-NotI fragment from pDT 18 and theNotI-XbaI fragment from pM7 into pCL1920 (SEQ ID NO:41). The glucoseisomerase promoter sequence from Streptomyces (SEQ ID NO:42) was clonedby PCR and inserted into EcoRI-HinDIII sites of pLitmus28 to constructpDT5. pCL1925 was constructed by inserting EcoRI-PvuII fragment of pDT5into the EcoRI-PvuII site of pCL1920. pDT24 was constructed by cloningthe HinDIII-MluII fragment of pDT21 and the MluI-XbaI fragment of pDT21into the HinDIII-XbaI sites of pCL1925.

Construction of an Expression Cassette for dhaT and dhaB(1,2,3,X):

pDT21 was constructed by inserting the SalI-NotI fragment from pDT 18and the NotI-XbaI fragment from pM7 into pCL1920 (SEQ ID NO:41). Theglucose isomerase promoter sequence from Streptomyces (SEQ ID NO:42) wascloned by PCR and inserted into EcoRI-HinDIII sites of pLitmus28 toconstruct pDT5. pCL1925 was constructed by inserting EcoRI-PvuIIfragment of pDT5 into the EcoRI-PvuI site of pCL1920. pDT24 wasconstructed by cloning the HinDIII-MluII fragment of pDT21 and theMluI-XbaI fragment of pDT21 into the HinDIII-XbaI sites of pCL1925.

Construction of an Expression Cassette for dhaR, orfY, dhaT, orfX, orfWand dhaB(1,2,3,X):

pDT29 was constructed by inserting the SacI-EcoRI fragment of pHK28-26into SacI-EcoRI sites of pCL1925.

Construction of an expression cassette for dhaR, orfY, orfX, orfW anddhaB(1,2,3,X):

A derivative of plasmid pDT29 was constructed in which all except thefirst 5 and the last 5 codons (plus stop codon) of the gene dhaT weredeleted by a technique known as PCR-mediated overlap extension. UsingpDT29 as template, 2 primary PCR products were generated using thefollowing primers:

SEQ ID NO:43 = 5′GAC GCA ACA GTA TTC CGT CGC3′; SEQ ID NO:44 = 5′ATG AGCTAT CGT ATG TTC CGC CAG GCA TTC TGA GTG TTA ACG3′; SEQ ID NO:45 = 5′GCCTGG CGG AAC ATA CGA TAG CTC ATA ATA TAC3′; SEQ ID NO:46 = 5′CGG GGC GCTGGG CCA GTA CTG3′.

SEQ ID NO:45 was paired with SEQ ID NO:46 to generate a product of 931bps and encompassing nucleic acid including 5′ dhaB1 (to unique ScaIsite), all of orfY, and the first five codons of dhaT. SEQ ID NO:43 waspaired with SEQ ID NO:44 to generate a product of 1348 bps andencompassing nucleic acid including the last five codons (plus stopcodon) of dhaT, all of orfX, all of orfW, and 5′ dhaR (to unique SapIsite). The 15 bases at the 5′ end of SEQ ID NO:44 constitute a tail thatis the inverse complement of a 15 base portion of SEQ ID NO:45.Similarly, the 11 bases at the 5′ end of SEQ ID NO:45 constitute a tailthat is the inverse complement of an 11 base portion of SEQ ID NO:44.Thus, the 2 primary PCR products were joined together after annealing(via 26 bp tail overlap) and extending by PCR, to generate a thirdnucleic acid product of 2253 bps. This third PCR product was digestedwith SapI and ScaI and ligated into pDT29 which was also digested withSapI and ScaI, to generate the plasmid pKP32, which is identical topDT29, except for the large, in-frame deletion within dhaT.

Example 7 Conversion of Glucose to 1,3-Propanediol Using E. Coli StrainKLP23/pAH48/pDT29 and the Improved Process Using KLP23/pAH48/pKP32Pre-Culture:

KLP23/pAH48/pDT29 and KLP23/pAH48/pKP32 were pre-cultured for seeding afermenter in 2YT medium (10 g/L yeast extract, 16 g/L tryptone, and 10g/L NaCl) containing 200 mg/L carbenicillin (or ampicillin) and 50 mg/Lspectinomycin. KLP23/pAH48/pKP32 is identical to KLP23/pAH48/pDT29except that dhaT is deleted.

Cultures were started from frozen stocks (10% DMSO as cryoprotectant) in500 mL of medium in a 2-L Erlenmeyer flask, grown at 35° C. in a shakerat 250 rpm until an OD₅₅₀ of approximately 1.0 AU was reached and usedto seed the fermenter.

Fermenter Medium:

The following components were sterilized together in the fermentervessel: 45 g KH₂PO₄, 12 g citric acid, 12 g MgSO₄.7H₂O, 30 g yeastextract, 2.0 g ferric ammonium citrate, 5 mL Mazu DF204 as antifoam, 1.2g CaCl₂.2H₂O, and 7.3 mL sulfuric acid. The pH was raised to 6.8 with20-28% NH₄OH and the following components were added: 1.2 gcarbenicillin or ampicillin, 0.30 g spectinomycin, 60 mL of a solutionof trace elements and glucose (from a 60-67 weight % feed). Afterinoculation, the volume was 6.0 L and the glucose concentration was 10g/L. The solution of trace elements contained (g/L): citric acid. H₂O(4.0), MnSO₄.H₂O (3.0), NaCl (1.0), FeSO₄.7H₂O (0.10), CoCl₂.6H₂O(0.10), ZnSO₄.7H₂O (0.10), CuSO₄.5H₂O (0.010), H₃BO₃ (0.010), andNa₂MoO₄.2H₂O (0.010).

Fermentation Growth:

A 15 L stirred tank fermenter was prepared with the medium describedabove. The temperature was controlled at 35° C. and aqueous ammonia(20-28 weight %) was used to control pH at 6.8. Initial values for airflow rate (set to minimum values of between 6 and 12 standard liters permin) and agitator speed (set to minimum values of between 350 and 690rpm) were set so that dissolved oxygen (DO) control was initiated whenOUR values reached approximately 140 mmol/L/h. Back pressure wascontrolled at 0.5 bar. DO control was set at 10%. Except for minorexcursions, glucose was maintained at between 0 g/L and 10 g/L with a60% or 67% (wt) feed. Vitamin B₁₂ or coenzyme B₁₂ was added as notedbelow.

Fermentation with KLP23/pAH48/pDT29:

A representative fermentation summary of the conversion of glucose to1,3-propanediol (1,3-PD) using E. coli strain KLP23/pAH48/pDT29 is givenin Table 4. Vitamin B₁₂ (0.075 g/L, 500 mL) was fed, starting 3 h afterinoculation, at a rate of 16 mL/h. The yield of 1,3-propanediol was 24wt % (g 1,3-propanediol/g glucose consumed) and a titer of 68 g/L1,3-propanediol was obtained.

TABLE 4 Representative fermentation summary of the conversion of glucoseto 1,3-propanediol (1,3-PD) using E. coli strain KLP23/pAH48/pDT29 TimeOD550 DO Glucose Glycerol 1,3-PD (h) (AU) (%) (g/L) (g/L) (g/L) 0 0 15012.9 0.0 0 6 17 80 8.3 3.1 1 12 42 53 2.8 12.5 9 18 98 9 5.7 12.6 32 24136 11 32.8 12.0 51 30 148 10 12.3 13.3 62 32 152 11 12.5 14.3 65 38 15911 1.5 17.2 68

Similar results were obtained with an identical vitamin B₁₂ feed attwice the concentration or bolus additions of vitamin B₁₂ across thetime course of the fermentation. The highest titer obtained was 77 g/L.

Improved Fermentation with KLP23/pAH48/pKP32:

A representative fermentation summary of the conversion of glucose to1,3-propanediol (1,3-PD) using E. coli strain KLP23/pAH48/pKP32 is givenin Table 5. Vitamin B₁₂ (0.150 g/L, 500 mL) was fed, starting 3 h afterinoculation, at a rate of 16 mL/h. After 36, h, approximately 2 L offermentation broth was purged in order to allow for the continuedaddition of glucose feed. The yield of 1,3-propanediol was 26 wt % (g1,3-propanediol/g glucose consumed) and a titer of 112 g/L1,3-propanediol was obtained.

TABLE 5 Representative fermentation summary of the improved conversionof glucose to 1,3-propanediol (1,3-PD) using E. coli strainKLP23/pAH48/pKP32 Time OD550 DO Glucose Glycerol 1,3-PD (h) (AU) (%)(g/L) (g/L) (g/L) 0 0 148 12.8 0.0 0 6 22 84 6.9 3.3 0 12 34 90 9.7 10.47 18 66 43 9.3 5.9 24 24 161 9 0.2 2.5 46 30 200 10 0.2 6.0 67 36 212 101.2 9.7 88 42 202 2 0.1 15.5 98 48 197 12 1.2 23.8 112

Similar results were obtained with an identical vitamin B₁₂ feed at halfthe concentration or bolus additions of vitamin B₁₂ across the timecourse of the fermentation. The highest titer obtained was 114 g/L.

Example 8 Engineering of Triosephosphate Isomerase Mutant of E. ColiKLP23 for Enhanced Yield of 1,3-Propanediol from Glucose

Construction of Plasmid for Triosephosphate Isomerase Gene Replacementin E. coli KLP23:

E. coli KLP23 genomic DNA was prepared using the Puregene DNA IsolationKit (Gentra Systems, Minneapolis, Minn.). A 1.0 kb DNA fragmentcontaining cdh and the 3′ end of triosephosphate isomerase (tpiA) geneswas amplified by PCR (Mullis and Faloona, Methods Enzymol. 155, 335-350(1987)) from KLP23 genomic DNA using primers SEQ ID NO:47 and SEQ IDNO:48. A 1.0 kb DNA fragment containing the 5′ end of tpiA, yiiQ, andthe 5′ end of yiiR genes was amplified by PCR from KLP23 genomic DNAusing primers SEQ ID NO:49 and SEQ ID NO:50. A ScaI site wasincorporated into primer SEQ ID NO:49. The 5′ end of primer SEQ ID NO:49was the reverse complement of primer SEQ ID NO:48 to enable subsequentoverlap extension PCR. The gene splicing by overlap extension technique(Horton et al., BioTechniques 8, 528-535 (1990)) was used to generate a2.0 kb fragment by PCR using the above two PCR fragments as templatesand primers SEQ ID NO:47 and SEQ ID NO:50. This fragment represented adeletion of 73% of the 768 bp tpiA structural gene. Overall, thisfragment had 1.0 kb flanking regions on either side of the ScaI cloningsite (within the partial tpiA) to allow for chromosomal gene replacementby homologous recombination.

The above blunt-ended 2.0 kb PCR fragment was cloned into the pCR-Bluntvector using the Zero Blunt PCR Cloning Kit (Invitrogen, San Diego,Calif.) to yield the 5.5 kb plasmid pRN106-2 containing kanamycin andZeocin resistance genes. The 1.2 kb HincII fragment from pLoxCat1(unpublished results), containing a chloramphenicol-resistance geneflanked by bacteriophage P1 loxP sites (Snaith et al., Gene 166, 173-174(1995)), was used to interrupt the tpiA fragment in plasmid pRN106-2 byligating it to ScaI-digested plasmid pRN106-2 to yield the 6.8 kbplasmid pRN107-1.

Engineering of Triosephosphate Isomerase Mutant RJ8m by Linear DNATransformation:

Using pRN107-1 as template and primers SEQ ID NO:47 and SEQ ID NO:50,the 3.2 kb fragment containing tpiA flanking regions and theloxP-CmR-loxP cassette was PCR amplified and gel-extracted. E. coliKLP23 was electrotransformed with up to 1 μg of this 3.2 kb linear DNAfragment and transformants that were chloramphenicol-resistant (12.5μg/mL) and kanamycin-sensitive (30 μg/mL) were further screened on M9minimal media for poor glucose utilization on 1 mM glucose, for normalgluconate utilization on 1 mM gluconate, and to ensure the glycerolnon-utilization phenotype of host KLP23 on 1 mM glycerol. An EcoRIdigest of genomic DNA from one such mutant, RJ8m, when probed with theintact tpiA gene via Southern analysis (Southern, J. Mol. Biol. 98,503-517 (1975)) indicated that it was a double-crossover integrant (tpiAgene replacement) since the two expected 6.6 kb and 3.0 kb bands wereobserved, owing to the presence of an additional EcoRI site within thechloramphenicol resistance gene. As expected, the host KLP23 andwild-type FM5 controls yielded single 8.9 kb and 9.4 kb bandsrespectively. This tpiA mutant was further analyzed by genomic PCR usingprimers SEQ ID NO:51 and SEQ ID NO:52, which yielded the expected 4.6 kbPCR fragment while for the same primer pair the host KLP23 and wild-typeFM5 strains both yielded the expected 3.9 kb PCR fragment. Whencell-free extracts from tpiA mutant RJ8m and host KLP23 were tested fortpiA activity using glyceraldehyde 3-phosphate as substrate, no activitywas observed with RJ8m. The tpiA mutant RJ8m was electrotransformed withplasmid pAH48 to allow glycerol production from glucose and also withboth plasmids pAH48 and pDT29 or pKP32 to allow 1,3-propanediolproduction from glucose. The chloramphenicol resistance marker waseliminated from RJ8m to give RJ8.

Example 9 Conversion of Glucose to 1,3-Propanediol Using E. Coli StrainRJ8/pAH48/pDT29 and the Improved Process Using RJ8/pAH48/pKP32Pre-Culture:

RJ8/pAH48/pDT29 and RJ8/pAH48/pKP32 were pre-cultured for seeding afermenter as described in Example 7. RJ8/pAH48/pKP32 is identical toRJ8/pAH48/pDT29 except that dhaT is deleted.

Fermenter Medium:

Fermenter medium was as described in Example 7.

Fermentation Growth:

Fermenter growth was as described in Example 7 except that initialvalues for air flow rate (set to minimum values of between 5 and 6standard liters per min) and agitator speed (set to minimum values ofbetween 300 and 690 rpm) were set so that dissolved oxygen (DO) controlwas initiated when OUR values reached between 60 and 100 mmol/L/h.Vitamin B₁₂ or coenzyme B₁₂ was added as noted below.

Fermentation with RJ8/pAH48/pDT29:

A representative fermentation summary of the conversion of glucose to1,3-propanediol (1,3-PD) using E. coli strain RJ8/pAH48/pDT29 is givenin Table 6. Vitamin B₁₂ was provided as bolus additions of 2, 16 and 16mg at 2, 8, and 26 h, respectively. The yield of 1,3-propanediol was 35wt % (g 1,3-propanediol/g glucose consumed) and a titer of 50.1 g/L1,3-propanediol was obtained.

TABLE 6 Representative fermentation summary of the conversion of glucoseto 1,3-propanediol (1,3-PD) using E. coli strain RJ8/pAH48/pDT29 TimeOD550 DO Glucose Glycerol 1,3-PD (h) (AU) (%) (g/L) (g/L) (g/L) 0 0 14010.6 0.1 0.0 6 5 107 11.1 0.5 0.4 10 16 90 8.5 1.7 1.3 14 25 86 1.8 2.45.9 19 38 53 3.5 5.9 15.4 25 53 38 0.1 9.2 26.7 31 54 10 4.5 7.4 39.0 3737 23 17.2 6.0 45.0 43 21 13 9.9 7.7 50.1Improved Fermentation with RJ8/pAH48/pKP32:

A representative fermentation summary of the conversion of glucose to1,3-propanediol (1,3-PD) using E. coli strain RJ8/pAH48/pKP32 is givenin Table 7. Vitamin B₁₂ was provided as bolus additions of 48 and 16 mgat approximately 26 and 44 hr, respectively. The yield of1,3-propanediol was 34 wt % (g 1,3-propanediol/g glucose consumed) and atiter of 129 g/L 1,3-propanediol was obtained.

TABLE 7 Representative fermentation summary of the improved conversionof glucose to 1,3-propanediol (1,3-PD) using E. coli strainRJ8/pAH48/pKP32. Time OD550 DO Glucose Glycerol 1,3-PD (h) (AU) (%)(g/L) (g/L) (g/L) 0 0 150 12.6 0.1 0 6 12 113 6.0 2.6 0 12 24 99 0.010.6 0 18 51 76 2.4 28.9 0 24 78 82 2.4 44.2 5 30 114 70 3.8 26.9 33 36111 72 0.0 20.0 57 42 139 65 0.1 21.9 69 48 157 36 0.1 22.4 79 55 158 250.2 21.4 94 64 169 14 0.1 15.8 113 72 169 12 0.1 13.4 119 74 162 14 0.114.8 129

Example 10 Identification of the E. Coli Non-Specific Catalytic Activity(yqhD) in the Improved 1,3-Propanediol Process

Demonstration of Non-Specific Catalytic Activity in1,3-Propanediol-Producing Fermentations with the Improved Catalyst:

A whole cell assay for 1,3-propanediol dehydrogenase activity was usedto demonstrate that the non-specific catalytic activity in E. coli ispresent under fermentative conditions after the addition of vitamin B₁₂and the production of 3-hydroxypropionaldehyde (3-HPA), but not before.A recombinant E. coli strain containing the glycerol-production and1,3-propanediol-production plasmids, pAH48 and pKP32, respectively, wasgrown in 10 L fermenters, essentially as described in Example 7, but inthe absence of vitamin B₁₂. A vitamin B₁₂ bolus (48 mg) was added whenthe tanks reached approximately 100 OD₅₅₀. Aliquots of cells were takenfrom the tanks immediately before and 2 h post-vitamin B₁₂ addition. Thecells were recovered by centrifugation and resuspended to their originalvolume in PBS buffer containing 150 μg/mL chloramphenicol to inhibit newprotein synthesis. An appropriate volume of the chloramphenicol treatedcells was added to 250 mL baffled flasks containing a reaction mixture(PBS buffer containing 10 g/L glucose, 10 g/L glycerol, 1 mg/L coenzymeB₁₂, and 150 μg/mL chloramphenicol) so that the final volume was 50 mLat an OD₅₅₀ of approximately 10. The flasks, protected from light, wereshaken at 250 rpm at 35° C. Aliquots for HPLC analysis were taken overtime. Time-dependent production of 3-HPA was observed in flaskscontaining cells recovered from the fermenter either pre- orpost-vitamin B₁₂ addition. In direct contrast, significant levels of1,3-propanediol were observed only in those flasks containing cellsrecovered from the fermenter post-vitamin B₁₂ addition.

Detection of Non-Specific Catalytic Activity in Cell-Free Extracts:

A native gel activity stain assay was used to demonstrate non-specificcatalytic activity in cell-free extracts. Cells were recovered, pre- andpost-vitamin B₁₂ addition, from representative 10-L fermentationsemploying recombinant E. coli strains containing the glycerol-productionand 1,3-propanediol-production plasmids, pAH48 and pKP32, respectively;and cell-free extracts were prepared by cell disruption using a Frenchpress. The cell-free extracts, a preparation of pure Klebsiellapneumoniae. 1,3-propanediol dehydrogenase (dhaT), and molecular weightstandards were applied to and run out on native gradient polyacrylamidegels. The gels were then exposed to either the substrates1,3-propanediol and NAD⁺ or ethanol and NAD⁺. As expected in the gelswhere 1,3-propanediol was the substrate, an activity stain for DhaT wasobserved which migrated on the native gel at approximately 340 Kdal.This activity was observed only in lanes where pure Klebsiellapneumoniae. 1,3-propanediol dehydrogenase was applied. In contrast,where 1,3-propanediol was the substrate and post-vitamin B₁₂ cell-freeextracts were applied, a non-specific catalytic activity was observed atapproximately 90 Kdal. When ethanol was used as a substrate, neither theDhaT band nor the non-specific catalytic activity band were visible, buta separate band was found pre- and post-vitamin B₁₂ addition atapproximately 120 Kdal. This new band most likely represents an alcoholdehydrogenase with specificity towards ethanol as substrate as istypically found in all organisms.

This native gel assay, where proteins are separated by molecular weightprior to the enzymatic assay step, offered greater sensitivity andaccuracy in measuring the reduction of 1,3-propanediol in thoseconstructs with low activity and where the activity is likely to bedistinct from the alcohol dehydrogenases with specificity towardsethanol as substrate that have been well characterized for E. coli andfound in all organisms. The dehydrogenase assay works on the principlethat dehydrogenase catalyzes the transfer of electrons from1,3-propanediol (or other alcohols) to NAD⁺. PMS (phenazinemethosulfate) then couples electron transfer between NADH and atetrazolium bromide dye (MTT,3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) whichforms a precipitate in the gel. After a few hours to overnight soakingin the substrates, the gels are washed to remove reagents and solubledye. At bands on the gel where there is an active dehydrogenase, aninsoluble blue dye forms. Various aspects of the assay have beendescribed by Johnson and Lin (J. Bacteriol. 169:2050 (1987)).

Purification and Identification of the Non-Specific Catalytic Activityin E. coli:

A large scale, partial purification of non-specific catalytic activitywas performed on cells harvested from the end of a typical1,3-propanediol production run as described in the improved processusing KLP23/pAH48/pKP32 of Example 7. The cell pellet (16 g) was washedand resuspended three times in 20 mL of 50 mM Hepes buffer, pH 7.5. Thecells in the suspension were lysed by sonication. The cell-free extractwas obtained by centrifugation (15 min, 20,000×g, 10° C.) and thesupernatant was further clarified by addition of 250 mg of protaminesulfate with stirring on ice. The supernatant obtained by centrifugation(20 min, 20,000×g, 10° C.) was fractionated by passage through aSuperdex 200 preparative grade column (6×60 cm) equilibrated with Hepesbuffer. Fractions of 10 mL each were collected and an aliquot of eachwas concentrated twenty five-fold using 10,000 MW cutoff Centricon®membranes prior to assay by the native gel activity stain. Thenon-specific catalytic activity was identified in fractions 107-112, andthe peak activity in fractions 108-109. A larger aliquot (7 mL each) offractions 108 and 109 were concentrated fifty-fold and loaded on alllanes of a 12-lane native gel. The gel was cut in half and one half wasstained for dehydrogenase activity where a dark blue band appeared thatrepresented the non-specific catalytic activity. The unstained gel wasaligned top to bottom with the stained gel and a band was cut on theunstained gel that corresponded to the band of non-specific catalyticactivity. The gel strip was pulverized and soluble protein was extractedby immersing the pulverized particles in 0.5 mL of 2D-loading buffer,heating to 95° C. for 5 min, and centrifugation to remove the gelparticles. The supernatant was loaded onto an isoelectricfocusing (IEF)strip for 2-dimension polyacrylamide gel electrophoresis (2D-PAGE) usingconditions described for 2D-PAGE of E. coli extracts in the Swiss 2Ddatabase (http://www.expasy.ch/ch2d/; Tonella et al. Electrophoresis19:1960-1971 (1998)). The gel was transferred to a PVDF membrane byelectroblotting. The membrane was stained for proteins using theColloidal blue gel stain. The stained blot used to obtain the identityof the non-specific catalytic activity is shown in FIG. 6. Spots wereidentified using standard techniques for amino terminus peptidesequencing. Only a single spot (Spot A) encoded for an oxidoreductaseactivity. Nineteen cycles of Spot A (FIG. 6) yielded a 100% identitymatch by the FASTA search tool with the amino-terminus of yqhD, an E.coli open reading frame with putative oxidoreductase activity. Completeamino acid sequence for the protein encoded by yqhD is given in SEQ IDNO:57; the corresponding DNA sequence is given in SEQ ID NO:58. The yqhDgene has 40% identity to the gene adhB in Clostridium, a probableNADH-dependent butanol dehydrogenase 2.

Gene Disruption of yqhD in E. coli KLP23:

Biochemical assays and amino-terminal amino acid sequencing suggestedthat non-specific catalytic activity may be encoded by the E. coli yqhDgene. This gene of unknown function encodes a hypotheticaloxidoreductase and contains two alcohol dehydrogenase signatures alsofound in the Citrobacter freundii and Klebsiella pneumoniae.1,3-propanediol dehydrogenase encoded by the dhaT gene.

To disrupt this gene, yqhD and 830 bp of 5′-flanking DNA sequence and906 bp of 3′-flanking DNA sequence were amplified from E. coli KLP23(Example 4) genomic DNA in a PCR using Taq polymerase and the followingprimers:

(SEQ ID NO:59) 5′-GCGGTACCGTTGCTCGACGCTCAGGTTTTCGG-3′ (SEQ ID NO:60)5′-GCGAGCTCGACGCTTGCCCTGATCGAGTTTTGC-3′The reaction was run at 94° C. for 1 min, 50° C. for 1 min, and 72° C.for 3 min for 35 cycles followed by a final extension at 72° C. for 5min. The resulting 3.7 Kb DNA fragment was purified, digested with SacIand KpnI and ligated to similarly digested pBluescriptII KS(+)(Strategene) for 16 h at 16° C. The ligated DNA was used to transform E.coli DH5α (Gibco/BRL) and the expected plasmid, pJSP29, was isolatedfrom a transformant demonstrating white colony color on LB agar (Difco)containing X-gal (40 μg/mL) and ampicillin (100 μg/mL). Plasmid pJSP29was digested with AflII and NdeI to liberate a 409 bp DNA fragmentcomprising 363 bp of the yqhD gene and 46 bp of 3′-flanking DNAsequence. The remaining 5,350 bp DNA fragment was purified and ligatedto the 1,374 bp AflII/NdeI DNA fragment containing the kanamycinresistance gene from pLoxKan2 (Genencor International, Palo Alto,Calif.) for 16 h at 16° C. The ligated DNA was used to transform E. coliDH5α and the expected plasmid, pJSP32-Blue, was isolated from atransformant selected on LB agar media containing kanamycin (50 μg/mL).Plasmid pJSP32-Blue was digested with KpnI and SacI and the 3,865 bpyqhD disruption cassette was purified and ligated to similarly digestedpGP704 (Miller and Mekalanos, J. Bacteriol. 170:2575-2583 (1988)) for 16h at 16° C. The ligated DNA was used to transform E. coli SY327 (Millerand Mekalanos, J. Bacteriol. 170:2575-2583 (1988)) and the expectedplasmid, pJSP32, was isolated from a transformant selected on LB agarmedia containing kanamycin (50 μg/mL). Plasmid pJSP32 was transformedinto E. coli KLP23 and transformants were selected on LB agar containingkanamycin (50 μg/mL). Of the 200 kanamycin-resistant transformantsscreened, two demonstrated the ampicillin-sensitive phenotype expectedfor a double-crossover recombination event resulting in replacement ofthe yqhD gene with the yqhD disruption cassette.

The disruption of the yqhD gene was confirmed by PCR using genomic DNAisolated from these two transformants as the template and the followingsets of primer pairs:

Set #1: (SEQ ID NO:61) ′-GCGAGCTCGACGCTTGCCCTGATCGAGTTTTGC-3′ (SEQ IDNO:62) 5′-CAGCTGGCAATTCCGGTTCG-3′ Set #2: (SEQ ID NO:63)5′-CCCAGCTGGCAATTCCGGTTCGCTTGCTGT-3′ (SEQ ID NO:64)5′-GGCGACCCGACGCTCCAGACGGAAGCTGGT-3′ Set #3: (SEQ ID NO:65)5′-CCGCAAGATTCACGGATGCATCGTGAAGGG-3′ (SEQ ID NO:66)5′-CGCCTTCTTGACGAGTTCTGAGCGGGA-3′ Set #4: (SEQ ID NO: 67)5′-GGAATTCATGAACAACTTTAATCTGCACAC-3′ (SEQ ID NO: 68)5′-GTTTGAGGCGTAAAAAGCTTAGCGGGCGGC-3′The reactions were run using either Expand High Fidelity Polymerase(Boehringer Manheim) or Platinum PCR Supermix containing Taq polymerase(Gibco/BRL) at 94° C. for 1 min, 50° C. for 1 min, and 72° C. for 2 minfor 35 cycles followed by a final extension at 72° C. for 5 min. Theresulting PCR products were analyzed by gel electrophoresis in 1.0%(w/v) agarose. The results summarized in Table 8 confirmed disruption ofthe yqhD gene in both transformants.

TABLE 8 Expected Size (bp) Primer Set yqhD disruption yqhD wild-typeObserved Size (bp) 1 1,200 no product ~1,200 2 1,266 no product ~1,266 32,594 no product ~2,594 4 no product 1,189 ~900The yqhD disruption deletes the 3′ end of yqhD, including 46 bp of3′-flanking intergenic DNA sequence. The deletion removes 363 bp of 3′yqhD coding sequence corresponding to 121 amino acids. A stop codon ispresent 15 bp downstream of the remaining yqhD coding sequence in thekanamycin resistance cassette.

Plasmids pAH48 and pKP32 were co-transformed into E. coli KLP23 (yqhD⁻)and transformants containing both plasmids were selected on LB agarcontaining ampicillin (100 μg/mL) and spectinomycin (50 μg/1 mL). Arepresentative transformant was tested for its ability to covert glucoseto 1,3-propanediol in 10 L fermentations either in the presence orabsence of vitamin B₁₂.

Demonstration that yqhD is Required for Significant 1,3-propanediolProduction in E. coli Strain KLP23/pAH48/pKP32:

Fermentations for the production of 1,3-propanediol were performed,essentially as described in Example 7, with the E. coli strain KLP23(yqhD⁻)/pAH48/pKP32 in order to test for the effect of the yqhDdisruption on 1,3-propanediol production.

A representative 10-L fermentation using the knockout of thenon-specific catalytic activity, E. coli strain KLP23(yqhD⁻)/pAH48/pKP32, is shown in Table 9. The organism steadilyaccumulated cell mass and glycerol until the addition of vitamin B₁₂when the OD₅₅₀ exceeded 30 A (10.4 h). Vitamin B₁₂ was added as a bolusaddition of 8 mg at 10.4 h and thereafter vitamin B₁₂ was continuouslyfed at a rate of 1.32 mg/h. In the 4 h that followed B₁₂ addition,glucose consumption slowed, the oxygen utilization rate dropped andthere was no further increase in optical density. Fermentation ofglucose ceased and the glucose concentration in the tank accumulated.The highest titer of 1,3-propanediol obtained was 0.41 g/L. The organismwas checked for its viability by plating a dilution series of the cellson agar plates containing ampicillin and spectinomycin. The plates wereincubated for 24 h in a 30° C. incubator. There were no viable colonieson the plate from the fermentation of E. coli KLP23 (yqhD⁻)/pAH48/pKP32,Table 11.

By contrast, the cell suspension from a control tank to which no vitaminB₁₂ was added continued to accrue cell mass and glycerol until the 10-Ltank was full due to the complete addition of the glucose feed solution(Table 10). An agar plate viability determination by dilution series ofthe cell suspension at the end of this fermentation showed a viable cellcount that was consistent with the total cell number estimated by theoptical density value (Table 11).

TABLE 9 Representative fermentation summary of the failed conversion ofglucose to 1,3-propanediol (1,3-PD) using E. coli strain KLP23(yqhD-)/pAH48/pKP32. time OD₅₅₀ DO glucose glycerol 1,3-PD (h) (AU) (%)(g/L) (g/L) (g/L) 0 0.4 150 11.3 0.05 0 2.3 3.0 134 10.7 0.13 0 4.3 10.885.0 8.2 1.41 0 8.3 23.1 81.8 0.9 10.0 0 16.3 37.2 149 13.1 21.4 0.4118.3 47.6 149 18.9 21.6 0.39 20.3 39.6 149 24.4 22.3 0.42 23.8 33.6 14925.4 22.0 0.41

TABLE 10 Representative fermentation summary of the conversion ofglucose to glycerol using E. coli strain KLP23 (yqhD−)/pAH48/pKP32. Time(h) OD₅₅₀ (AU) DO (%) glucose (g/L) glycerol (g/L) 0 0.2 148 9.5 0.062.2 2.8 128 8.9 0.13 4.2 10.4 58.5 7.0 1.4 8.2 21.6 57.6 2.7 11.2 16.276.8 10.7 0 40.5 20.2 117 10.2 0 52.9 23.7 154 8.5 0 63.9 36.2 239 10.10.1 122

TABLE 11 Representative summary of viability plate counts from endpointsof fermentations of glucose using E. coli strainKLP23(yqhD⁻)/pAH48/pKP32 in the absence and presence of vitamin B₁₂.vitamin B₁₂ time (h) at endpoint OD₅₅₀ (AU) viable counts (cfu/mL) no36.2 239 2.1E11 yes 23.8 33.6 0 yes 23.8 41.2 0

1-6. (canceled)
 7. A recombinant microorganism comprising genes encodinga G3PDH, a G3P phosphtase, a dehydratase, and a dehydratase reactivationfactor wherein no functional dhaT gene encoding a 1,3 propanedioloxidoreductase activity is present in the recombinant microorganism andthe microorganism is selected from the group consisting of Citrobacter,Enterobacter, Clostridium, Klebsiella, Aerobacter, Lactobacillus,Aspergillus, Saccharomyces, Schizosaccharomyces, Zygosaccharomyces,Pichia, Kluyveromyces, Candida, Hansenula, Debaryomyces, Mucor,Torulopsis, Methylobacter, Salmonella, Bacillus, Aerobacter,Streptomyces and Pseudomonas. 8-10. (canceled)
 11. The recombinantmicroorganism of claim 7, further comprising a set of endogenous genes,each having a mutation inactivating the gene, the set consisting of: (a)a first gene encoding a polypeptide having glycerol kinase activity; (b)a second gene encoding a polypeptide having glycerol dehydrogenaseactivity; and (c) a third gene encoding a polypeptide havingtriosephosphate isomerase activity. 12-17. (canceled)
 18. A recombinantE. coli comprising: a set of exogenous genes consisting of: (i) at leastone gene encoding a polypeptide having a dehydratase activity; (ii) atleast one gene encoding a polypeptide having glycerol 3-phosphatedehydrogenase activity; (iii) at least one gene encoding a polypeptidehaving glycerol 3-phosphatase activity; and (iv) at least one geneencoding a dehydratase reactivation factor; wherein no functional dhaTgene encoding a 1,3 propanediol oxidoreductase activity is present inthe recombinant E. coli.
 19. A recombinant E. coli comprising: a set ofexogenous genes consisting of (i) at least one gene encoding apolypeptide having glycerol 3-phosphate dehydrogenase activity; (ii) atleast one gene encoding a polypeptide having glycerol 3-phosphataseactivity; and (iii) at least one subset of genes encoding the geneproducts of dhaR, orfY, orfX, orfW, dhaB1, dhaB2, dhaB3 and orfZ,wherein no functional dhaT gene encoding a 1,3 propanedioloxidoreductase activity is present in the recombinant E. coli.
 20. Therecombinant E. coli of claim 19 further comprising a set of endogenousgenes, each gene having a mutation inactivating the gene, the setconsisting of: (a) a gene encoding a polypeptide having glycerol kinaseactivity; (b) a gene encoding a polypeptide having glyceroldehydrogenase activity; and (c) a gene encoding a polypeptide havingtriosephosphate isomerase activity. 21-29. (canceled)